Drosophilid flies feed on various substances such as fermenting fruits, flowers, leaves, and mushrooms. Among these, most of the mushroom-feeding species belong to the Zygothrica genus group or the Drosophila quinaria species group. In addition, some mycophagous Drosophila species have a tolerance to α-amanitin, a major toxin of lethal Amanita mushrooms. Previous studies have suggested that the α-amanitin tolerance is tightly correlated with the evolution of mycophagy in drosophilid flies. To test the hypothesis, we investigated the α-amanitin tolerance in adult flies of 16 species, including both mycophagous and non-mycophagous species. The results showed that all the mycophagous species investigated were tolerant to α-amanitin, while the non-mycophagous species were not tolerant with two exceptions, generalists D. hydei and D. sternopleuralis. This suggests that the character of α-amanitin tolerance has been acquired more or less independently from the food-habit evolution in the family Drosophilidae.