Incipient speciation between stocks of Sub-Saharan Africa and cosmopolitan populations in D. melanogaster has been reported for decades but genes involved in their mating preference are elusive because prevalence of inversions in African populations hampers detailed genetic analyses. We employed a new strategy to explore the variations in a cosmopolitan population and confirmed 42 genes which affected significantly on the copulation success of cosmopolitan males to Zimbabwean Z30 females. Surprisingly, most of the RNAi mediated disruptions of these genes enhanced the male copulation success and a weak driver did so rather than the strong ones. Using mutants affecting female’s sensory systems and surgical removal of female antennae, we demonstrated that the effects of gene-disruption on cosmopolitan males were perceived by Ir8a/Ir25a mediated olfaction. We further specified male oenocytes as the target of these genes and most of methyl branching cuticular hydrocarbons were reduced in the gene-disrupted males.