Drosophila bipectinata and D. malerkotliana are two closely related species that share common ecological niches throughout their distribution zone, which comes under the Oriental-Australian zoogeographical regions. These two species have been found to share several common genetic characteristics, and due to this, they may experience interspecific mating under laboratory conditions and produce hybrid progeny. The population genetic work on these two species has been inadequately done by considering inversions and enzyme polymorphisms. We decided to consider the genetic polymorphism involving commonly persistent chromosomal inversions, allozymes, and microsatellite variants of the two species to envisage genetic differentiation among the natural populations of these two species sampled from distant localities of Indian cities. The results of this study indicate that the Indian populations of both species are genetically structured. There exists graded variation (clinal variation) in the level of heterozygosity from north to south, as an increase in the observed heterozygosity prevailed from north to south. This trend was observed in the populations of both species, which hints towards similar genetic changes being experienced by their members all along their distribution area. The phylogenetic trees based on the extent of genetic identity between the paired populations of these two species portray two distinct clusters, one for the two populations of the north and the other for the remaining populations of the south. Further, through this study, it can be stated with certainty that there exists ‘isolation by distance’ as the north and south populations of both species genetically significantly vary from each other.