<p>Two populations of Drosophila ananassae from Mysore (the Indian subcontinent) and Port Blair (the Andaman Islands) offer a unique opportunity to explore evolutionary divergence driven by genetic and environmental factors. In this study, we investigated the divergence using an integrative approach that combined morphometric, cuticular hydrocarbon profiling, behavioral, and genetic analyses. Our results revealed significant differences between the two populations in their morphometric traits, in mating strategies, distinct hydrocarbon signatures and DNA barcodes. The heterotypic reciprocal crosses between the two populations exhibited mating incompatibility, resulting in failure to produce viable progeny. Mitochondrial COI sequences revealed that these two populations constitute two different clades with proximity to the reference sequences of <i>D. ananassae</i>. Considering all these attributes together, we infer that the <i>D. ananassae</i> populations from Mysore and Post Blair are at intermediate stage of speciation under different ecological exposure and that revealed divergence patterns often associated with semi-species within the ananassae species complex.</p>