Abstract Low desiccation resistance of Drosophila ananassae reflects its rarity outside the humid tropics. However, the ability of this sensitive species to evolve under seasonally varying subtropical areas is largely unknown. D. ananassae flies are mostly lighter during the rainy season but darker and lighter flies occur in the autumn season in northern India. We tested the hypothesis whether seasonally varying alternative body color phenotypes of D. ananassae vary in their levels of environmental stress tolerances and mating behavior. Thus, we investigated D. ananassae flies collected during rainy and autumn seasons for changes in body melanization and their genetic basis, desiccation-related traits, cold tolerance and mating propensity. On the basis of genetic crosses, we found total body color dimorphism consistent with a single gene model in both sexes of D. ananassae. A significant increase in the frequency of the dark morph was observed during the drier autumn season, and body color phenotypes showed significant deviations from Hardy-Weinberg equilibrium, which suggests climatic selection plays a role. Resistance to desiccation as well as cold stress were two- to three-fold higher in the dark body color strain as compared with the light strain. On the basis of no-choice mating experiments, we observed significantly higher assortative matings between dark morphs under desiccation or cold stress, and between light morphs under hot or higher humidity conditions. To the best of our knowledge, this is the first report on the ecological significance of seasonally varying total body color dimorphism in a tropical species, D. ananassae.