Misgurnus anguillicaudatus and Paramisgurnus dabryanus hybrids were generated via full diallel crosses between the Meishan (M_M) and Fuyang (M_F) populations of M. anguillicaudatus and Meishan (P_M) and Ziyang (P_Z) populations of P. dabryanus. The purebred and hybrid offspring were co-cultured in a pool for 4 months, and 96.5% of the offspring could be assigned to a single parental pair based on nine potentially cross-species polymorphic microsatellites. The average body weight (BW) of the P_M × P_Z (female × male, in this order hereafter) hybrids was the highest (4.88 g), followed by that of the P_Z × P_M and P_M × P_M hybrids. The general combining abilities (GCA) of BW from both the sires and dams of P_M and P_Z were high, and that from M_M and M_F were low. The specific combining ability (SCA) of P_M × P_Z was the largest (16.61), followed by that of P_Z × P_M, M_F × M_M, and M_M × M_F (−15.89). The combination of M_F × P_M, M_F × P_Z, and P_M × M_F showed > 10% heterosis. This is the first study to use microsatellite-based parentage assignment in hybrid fish and lays foundation for loach crossbreeding.