At the beginning of the voyage of the HMS Beagle, Darwin was almost incapacitated with nausea. He swung miserably in his hammock in the small cabin he shared with several of the ship's officers or hung by the rail of the ship. Eventually, the nausea passed away and he was able to focus on the voyage itself.

The ship's first stop was meant to be Tenerife in the Canary Islands, the same place that Darwin had hoped to visit with Henslow. Unfortunately, because of a recent cholera outbreak in England they would have been quarantined for twelve days before landing, so Captain FitzRoy gave the order to set sail for St. Jago in the Cape Verde Islands, 300 miles off the African coast. Along the way, Darwin began his work as a naturalist by collecting plankton. When they landed at St. Jago he hiked through the volcano hills, encountering his first tropical jungle in a small valley and seeing real evidence of geological change: a layer of compressed sea shells in the cliffs thirty feet above sea level. Leaving St. Jago on February 8, 1831, they stopped at St. Paul's Rocks to kill birds for food, then crossed the Equator on February 16.

They reached South America at Bahia, at All Saints' Bay, on February 28. They spent several weeks there before departing for Rio on March 18. Upon arriving, on April 5, Darwin received letters from home for the first time since leaving England. He was surprised to learn that Fanny Owen, who had just broken off an engagement before he left, was now married to another man. While the Beagle surveyed the coastline, Darwin explored the interior, riding with gauchos into the Brazilian jungle from a home base in a rented cottage on Botafogo Bay. He starting filling books with notes on the flora, fauna, and geological formations he encountered. He hunted and collected, setting aside samples to be sent to Henslow in England. On July 7, they continued south to Montevideo. For the next few months the Beagle surveyed up and down the coast while Darwin explored on land. His most exciting find was a fossil megatherium, an extinct ground-dwelling relative of the sloth, discovered in a cliff face at Punta Alta on September 22. Somewhere in the back and forth between Montevideo and Buenos Aires, Darwin found time to assemble his first box of samples and send it off to Henslow.

On December 18, 1832, they finally set sail for Tierra del Fuego at the southern tip of South America. There Darwin was shocked to meet the native people, the Fuegians, who seemed to him to be hardly human. In January 1833, on their way to the western coast of Tierra del Fuego, they were almost sunk by bad weather and enormous swells. But they made it safely to the home territory of the Fuegians they had brought on board from England, two men and a woman who had been taken hostage by FitzRoy on a previous trip. They dropped off the Fuegians along with a British missionary who hoped to spread Christianity among the 'heathen' Fuegians. But when the Beagle returned nine days later all of the missionary's belongings had been stolen and the shaken missionary asked to be taken back on board. The next few months saw the Beagle return to the east coast of South America, stopping by the Falkland Islands of the coast of Argentina and then returning to Montevideo. While a second ship that FitzRoy had bought in the Falklands was being refitted for the journey around the Horn, Darwin headed off into the interior, traveling 200 miles in two weeks and killing eighty different kinds of birds, as well as other species, along the way. He continued to send his samples to Henslow.

The Beagle finally headed south again in December 1833, passing Port Desire and Port St. Julian on its way through the Straits of Magellan to where the hostage Fuegians had been dropped off months before. There they found that all the new habits taught the Fuegians in England had worn off: they were back to living as they had before. Then, once again, the Beagle returned to the Falklands and Montevideo for another survey of the coast. Darwin headed inland towards the Andes with a group of men and supplies, but provisions ran low and they were forced to turn back before reaching them. Fortunately Darwin knew he would have a chance to reach them from the other side when the Beagle went to Chile.

They finally made it around the Horn and arrived at the island of Chiloé, off the west coast of southern South America, on June 6, 1834. From there they went to Valparaiso on July 23. Since it was winter, it was too dangerous to reach the Andes proper, but Darwin made it to the foothills in August, returning through Santiago. There was a brief scare when he returned: FitzRoy had apparently had a breakdown because of doubts about the accuracy of his measurements on the eastern coast of South America, and had resigned his captainship. Fortunately he officers convinced him to resume his post and it was resolved that there was no need to return to the east coast for further measurements. In February 1835, Darwin experienced an enormous earthquake that left local cities in ruins. In March, he finally achieved his dream of seeing the Andes up close, having received some financial help for the expedition from his father, Robert.

After returning from the successful Andes expedition Darwin rejoined the Beagle for the trip north to Lima, where they arrived on July 19, 1835. Unfortunately, political unrest was such that they were confined to the port, unable to enter the city itself. They finally headed west into the Pacific two months later, catching their first glimpse of the Galapagos Islands, which Darwin was later to make famous, on September 15.