Abstract:To identify the optimal internal reference genes for qRT-PCR analysis in loach (Misgurnus anguillicaudatus), six commonly used reference genes, including ACTB, TUBA, EF1A, GAPDH,TUBB and 18S rRNA were selected as candidates, and their expression stability during different developmental stages, acrosstissues and betweenploidy were evaluated by using BestKeeper, geNorm, NormFinder, ΔCt and RefFinfer software. The results showed that, during the process of embryonic development, the expression levels of TUBB and TUBA were more stable than the other genes, suggesting that they can be used as reference genes. Besides, the expression level of TUBB showed no significant difference between diploids and tetraploids (P>0.05), suggesting it is the most suitable internal reference genes for qRT-PCR analysis during embryonic development between ploidy. Among different tissues of diploids and tetraploids, the expression levels of ACTB and 18S rRNA were more stable than the other genes, thus they can be used as internal control genes. Of which,18S rRNA was considered as the most suitable internal control genes, because the expression level of it exhibited no significant difference in different tissues between diploids and tetraploids (P>0.05).