Drosophila nasuta nasuta and Drosophila nasuta albomicans are cross-fertile races of Drosophila. Hybridization between these races in the laboratory has given rise to new races (Cytoraces), among which karyotypic composition differs from one another and also from those of the parental races. In this study, we search for the evidence of incipient reproductive isolation among the parental races and four Cytoraces by assessing the fraction of no-matings, mating latency and copulation duration in all possible types of homo- and heterogamic crosses (N = 4184). In no-choice conditions, the latency time (time to initiation of copulation) is lower in homogamic crosses than in heterogamic crosses for both parental races and Cytoraces. Latency time and copulation duration are negatively correlated, whereas fraction of no matings is positively correlated with latency time. Thus these six closely related races of the nasuta-albomicans complex show the initiation of the earliest stages of pre-zygotic isolation, manifested as a tendency for matings to be initiated earlier and more often, and for a longer duration, among homogamic rather than heterogamic individuals