This study was conducted to identify taxonomic differences in the characteristics of Acheilognathus majusculus and A. yamatsutae during their initial life history via an interspecific hybridization experiment. Hatching time required 36 h for MY and 49 h for YM at 21.5°C, showing a significant difference of 13 h between the hybrids. The hatching rates of the cross-bred eggs were 30% for cross MY (A. majusculus♀×A. yamatsutae♂) and 40% for cross YM (A. yamatsutae♀×A. majusculus♂). The hatching larvae size was total length 3.13-3.43 mm in MY and total length 3.89- 4.22 mm in YM, which was larger in YM. The hybridization test between A. yamatsutae and A. majusculus that live in the same water stream confirmed that no interspecific reproductive isolation occurred.