The cactophilic species Drosophila serido Vilela and Sene 1977 and Drosophila antonietae Tidon-Sklorz and Sene 2001 are two closely related species that have partially overlapping distributions in the Florianópolis-SC locality on the southern Brazilian coast. To characterize this contact zone with respect to possible introgression events and the differential use of cacti hosts, we analyzed male individuals that were collected in their natural environment or from the necrotic cacti in which they were reared, by using aedeagus morphology, isocitrate dehydrogenase isozymes, and the mitochondrial gene cytochrome oxidase I as diagnostic markers. Morphological and molecular markers showed a bimodal distribution, although there was some incongruence, suggesting the presence of asymmetric introgression events. These results suggest that gene flow occurred in the past but then ceased, possibly due to reinforcement of prezygotic reproductive isolation. Analysis of individuals reared in necrotic cacti of two sympatric species in this region showed that D. antonietae, but not D. serido, preferentially uses Cereus hildmannianus Mill. as a cactus host, which may represent an important influence on ecological differentiation by affecting the dynamics of these species in this area of sympatry.