First officer of the Starship Voyager and former member of the Maquis resistance group. Chakotay was of Native American descent and fiercely proud of his ancestry. As a child, however, Chakotay rebelled against his heritage. At the age of 15, Chakotay traveled with his father, Kolopak, to Earth in search of the ancient Rubber Tree People. During the expedition, Chakotay hurt his father deeply by announcing that he was leaving his people to attend Starfleet Academy. Kolopak accepted his son's rebellion, noting that Chakotay had been a breech birth, indicating a problem child. Chakotay's application to the academy had been sponsored by Captain Sulu. After his father's death, Chakotay came to recognize the importance of his people's heritage, and he tattooed his forehead in honor of his father and their ancestors. One of his ancestors was a school teacher in Arizona on Earth in the 20th century.

During Chakotay's first year at Starfleet Academy, he trained as a pilot over Earth's North American continent and spent a couple of months on Venus learning how to handle atmospheric storms. He later learned to dodge asteroids in that system's asteroid belt. Chakotay had an interest in archaeology. After Chakotay graduated from the academy, he was a member of the Starfleet team that made first contact with the Tarkannans. He later left Starfleet to join the Maquis in defense of his homeworld against the Cardassians. He commanded a Maquis ship that was lost in the Badlands in 2371 while fleeing from a Cardassian ship. Chakotay and his crew were swept into the distant Delta Quadrant by the Caretaker, where they were trapped when thier ship was destroyed by the Kazon-Ogla. Chakotay and his fellow Maquis subsequently accepted an invitation to join the crew of the Starship Voyager under the command of Captain Kathryn Janeway. Under this arrangement, Chakotay became the ship's first officer, replacing Lieutenant Commander Cavit. The difficult conditions in the Delta Quadrant led Chakotay to question whether Starfleet's idealism was appropriate in this distant part of the galaxy, wondering if the expediency of Maquis techniques might be wiser.

Chakotay practiced his people's vision quest rituals, seeking direction from his animal guide. He used his medicine bundle to help invoke these rituals. Chakotay would occasionally help those close to him experience the vision quest in search of their own animal guides. Chakotay honored his people's traditional medical practices, including the use of a medicine wheel to help guide his spirit back to his body when it was displaced by trianic energy beings on stardate 48734.

While in the Maquis, Chakotay was romantically involved with Seska, unaware that she was a Cardassian agent who had been surgically altered to appear Bajoran. Seska, who later defected to the Kazon, subsequently deceived Chakotay, telling him that she had impregnated herself with a sample of his DNA.

Chakotay became afflicted wiht a potentially fatal viral disease in 2372 after exposure to an insectoid life-form on a planet in the Delta Quadrant. Extensive research determined that the condition could remain benign as long as he remained on the planet, but that he could not survive if he left. Voyager Captain Kathryn Janeway was also striken with the disease. Determined that her people should not sacrifice thier chance to return home, Janeway ordered her ship to continue their voyage, leaving Chakotay and Janeway behind. On the planet, Chakotay's wilderness skills that he learned as a boy from Kolopak, his father, proved invaluable. After several weeks, Voyager's crew, with the aid of Vidiian physician Danara Pel, was able to obtain an antiviral medication, and return to successfully treat Chakotay and Janeway. Chakotay and Janeway grew closer during their time alone together on the planet.

In 2373, Chakotay was temporarily assimilated into a Borg collective while on a mission of exploration into the Nekrit Expanse. His brief exposure to the Borg group consciousness helped heal him from a serious injury. Chakotay later assisted a group of former Borg drones to reestablish a group consciousness, which they felt necessary to a life of harmony.

In 2373, following the discovery that Voyager was under observation by a race known as the Voth, Chakotay was taken prisoner by a Voth scientist Professor Gegen. It was later learned that the Voth scientists had found the bones of Ensign Hogan on Hanon IV and discovered that the Voth shared 47 genetic markers with humans, making very probable that the two were distantly related genetically. Chakotay was taken by Gegen to provide proof of a 'Distant Origin theory' that Gegen had been working on for many years. Gegen later released Chakotay following the capture of Voyager by the Voth. Following a 'trial' in which Gegen was forced to renounce his theory the Voyager and crew were released with a warning not to enter Voth space.

On stardate 51082, while on a survey mission, Chakotay's shuttle was shot down when he stopped to investigate some Omicron radiation readings from a nearby planet. Chakotay was subsequently brainwashed by a race known as the Vori and impressed into their army. Chakotay was later found by Tuvok, with the help of the Vori's enemies the Kradin, and returned to Voyager.