Many mycophagous Drosophila species have adapted to tolerate high concentrations of mycotoxins, an ability not reported in any other eukaryotes. Although an association between mycophagy and mycotoxin tolerance has been established in many Drosophila species, the genetic mechanisms of the tolerance are unknown. This study presents the inter- and intraspecific variation in the mycotoxin tolerance trait. We studied the mycotoxin tolerance in four Drosophila species from four separate clades within the immigrans-tripunctata radiation from two distinct locations. The effect of mycotoxin treatment on 20 isofemale lines per species was studied using seven gross phenotypes: survival to pupation, survival to eclosion, development time to pupation and eclosion, thorax length, fecundity, and longevity. We observed interspecific variation among four species, with D. falleni being the most tolerant, followed by D. recens, D. neotestacea, and D. tripunctata, in that order. The results also revealed geographical variation and intraspecific genetic variation in mycotoxin tolerance. This report provides the foundation for further delineating the genetic mechanisms of the mycotoxin tolerance trait.