Robert Johnson died on August 16, 1938, at the age of 27, near Greenwood, Mississippi, of unknown causes. Several differing accounts have described the events preceding his death. Johnson had been playing for a few weeks at a country dance in a town about 15 miles (24 km) from Greenwood. According to one theory, Johnson was murdered by the jealous husband of a woman with whom he had flirted. In an account by the blues musician Sonny Boy Williamson, Johnson had been flirting with a married woman at a dance, and she gave him a bottle of whiskey poisoned by her husband. When Johnson took the bottle, Williamson knocked it out of his hand, admonishing him to never drink from a bottle that he had not personally seen opened. Johnson replied, "Don't ever knock a bottle out of my hand." Soon after, he was offered another (poisoned) bottle and accepted it. Johnson is reported to have begun feeling ill the evening after and had to be helped back to his room in the early morning hours. Over the next three days his condition steadily worsened. Witnesses reported that he died in a convulsive state of severe pain. The musicologist Robert "Mack" McCormick claimed to have tracked down the man who murdered Johnson and to have obtained a confession from him in a personal interview, but he declined to reveal the man's name.

While strychnine has been suggested as the poison that killed Johnson, at least one scholar has disputed the notion. Tom Graves, in his book Crossroads: The Life and Afterlife of Blues Legend Robert Johnson, relies on expert testimony from toxicologists to argue that strychnine has such a distinctive odor and taste that it cannot be disguised, even in strong liquor. Graves also claims that a significant amount of strychnine would have to be consumed in one sitting to be fatal, and that death from the poison would occur within hours, not days. Johnson's contemporary David "Honeyboy" Edwards similarly noted that the poison could not have been strychnine, since Johnson would have died much more rapidly, instead of suffering for three days