Behavioral isolation in animals can be mediated by inherent mating preferences and assortative traits, such as divergence in the diel timing of mating activity. Although divergence in the diel mating time could, in principle, promote the reproductive isolation of sympatric, conspecific populations, there is currently no unequivocal evidence of this. We conducted different mate-choice experiments to investigate the contribution of differences in diel mating activity to the reproductive isolation of the rice and water-oat populations of Chilo suppressalis. The results show that inter-population difference in diel mating activity contributes to assortative mating in these populations. In the rice population, most mating activity occurred during the first half of the scotophase, whereas in the water-oat population virtually all mating activity was confined to the second half of the scotophase. However, when the photoperiod of individuals from the water-oat population was altered to more closely align their mating activity with that of the rice population, mate choice was random. We conclude that inter-population differences in diel mating time contribute to assortative mating, and thereby the partial reproductive isolation, of these host-associated populations of C. suppressalis.