In light of reproductive isolation being a fundamental aspect of the biological species concept, we performed crossing experiments using six species from three genera (Hoplobatrachus, Euphlyctis and Fejervarya) of family Dicroglossidae to explore postmating isolation in dicroglossid frogs. Our results revealed gametic isolation among these genera, although the intergeneric hybrids between female E. cyanophlyctis and male H. chinensis were not viable at the tadpole stage, while the hybrids between female E. cyanophlyctis and male H. tigerinus were inviable at the hatching stage. These results showed complete hybrid inviability between the two genera. Almost all interspecific hybrids between female H. tigerinus and male H. chinensis died of underdevelopment at the tadpole stage, whereas several hybrids developed normally and survived to maturity. Chromosomal observations and mtDNA and allozyme analyses confirmed that these mature hybrids were allotriploid, with two maternal genomes and one paternal genome. The present results suggest that the allotriploids were produced spontaneously, and histological observations confirmed their sex as sterile males. We also investigated the molecular relationships between H. tigerinus, H. chinensis, and the interspecific allotriploids by mitochondrial Cytb, 12S and 16S rRNA gene analyses. The maternal inheritance mode of mitochondrial genomes was retained in the hybrids. Finally, the present results suggest that the degree of postmating isolation reflects phylogenetic relationship. In addition, we speculate that allotriploids may be produced via hybridization among cryptic species.