Similar phenotypic changes have evolved independently in many animal taxa. It is unknown whether independent changes involve the same or different developmental and genetic mechanisms. Myriad pigment patterns in the genusDrosophilaoffer numerous opportunities to address this question. Previous studies identified regulatory and structural genes involved in the development and diversification of pigmentation in selected species. Here, we examineDrosophila americanaandDrosophila novamexicana, interfertile species that have evolved dramatic pigmentation differences during the few million years since their divergence. Interspecific genetic analysis was used to investigate the contribution of five specific candidate genes and other genomic regions to phenotypic divergence by testing for associations between molecular markers and pigmentation. At least four distinct genomic regions contributed to pigmentation differences, one of which included theebonygene. Ebony protein was expressed at higher levels in the more yellowD. novamexicanathan the heavily melanizedD. americana. Because Ebony promotes yellow pigment formation and suppresses melanization, the expression difference and genetic association suggest that evolution at theebonylocus contributed to pigmentation divergence betweenD. americanaandD. novamexicana. Surprisingly, no genetic association with theyellowlocus was detected in this study, and Yellow expression was identical in the two species. Evolution at theyellowlocus underlies pigmentation divergence among otherDrosophilaspecies; thus, similar pigment patterns have evolved through regulatory changes in different genes in different lineages. These findings bear upon understanding classic models of melanism and mimicry.