Matsumuraeses phaseoli and M. falcana cannot be classified with the external morphological characters. Although differences in the morphology of male genitalia and in the mitochondrial DNA sequence of cytochrome oxidase I between the two species have been found, there is no information that the two species are biologically different species till now. We, therefore in this study, tried to cross the two species to observe postzygotic incompatibility in the next hybrid generations and to know whether the two species are 'biological species' or not. In crossing between the parents, two kinds of F1 hybrids were produced successfully. In inter- and intra-breeding between F1 hybrids, two lines crossed with females of F1 hybrid produced by females of M. phaseoli could not produce F2 adults to show inviability of larvae. The other two lines produced F2 adults successfully and the F2 adults produced F3 larvae in inbreeding. In back-crossing between parents and F1 hybrids, the two lines of 8 breeding lines, in which females of F1 hybrid were produced by females of M. phaseoli, could not produce the next generation of larvae. The other six lines produced F2 adults successfully. The results indicated that maternal factors of F1 hybrid produced by M. phaseoli females contributed to create the incompatibility between the F1 females and other lines. In conclusion, the results showed a postzygotic reproductive isolation between M. phaseoli and M. falcana in part.