Pigmentation is a rapidly evolving trait that can play important roles in mimicry, sexual selection, thermoregulation, and other adaptive processes in many groups of animals. In Drosophila, pigmentation can differ dramatically among closely related taxa, presenting a good opportunity to dissect the genetic changes underlying species divergence. In this report, we investigate the genetic basis of color pattern variation between two allopatric subspecies of Drosophila malerkotliana, a widespread member of the ananassae species subgroup. In D. malerkotliana malerkotliana, the last three abdominal segments are darkly pigmented in males but not in females, while in D. malerkotliana pallens both sexes lack dark pigmentation. Composite interval mapping in F2 hybrid progeny shows that this difference is largely controlled by three quantitative trait loci (QTL) located on the 2L chromosome arm, which is homologous to the 3R of D. melanogaster (Muller element E). Using highly recombinant introgression strains produced by repeated backcrossing and phenotypic selection, we show that these QTL do not correspond to any of the candidate genes known to be involved in pigment patterning and synthesis in Drosophila. These results, in combination with similar analyses in other Drosophila species, indicate that different genetic and molecular changes are responsible for the evolution of similar phenotypic traits in different lineages. This feature makes Drosophila color patterns a powerful model for investigating how the genetic basis of trait evolution is influenced by the intrinsic organization of regulatory pathways controlling the development of these traits.