9 missense mutation sites of body color related pathway genes were sequenced and investigated. In three small sample populations of Cynoglossus semilaevis with hyperpigmentation, pseudoalbino or normal body color, it was found that three mutation sites are significant correlation with pseudoalbino. Further verification by expanding samples showed that the C/T site at position 160 of type B endothelin receptor (ertB-160-C/T) was extremely significantly correlated with pseudoalbino. The G/A site at position 473 of tyrosinase (tyr-473-G/A) and the C/T site at position 1 178 of arachidonic acid 5-lipoxygenase (alox5-1178-C/T) were significantly correlated with pseudoalbino.The three mutation sites were located in the protein periplasmic-Binding-Protein-Type 1, tyosinase C-terminal and lipoxygenase domains. RT-qPCR assay revealed that:There was no evident difference in ertB expression in different colored areas of the skin of albino half-smooth tongue sole on the eyed side, however, the expression of ertB in the skin on the eyeless side was prominently higher than that in the skin on the eyed side,and the expression level of tyr in both pigmented and non-pigmented parts of the eyed side were remarkably higher than those of the eyeless side; The expression level of alox5 on the eyeless side was markedly higher than that on the eyed side; The alox5 expression in the albino area on the eyed side was prominently higher than that in the pigmented area on the eyed side. The mutation sites related to pseudoalbino of C. semilaevis cultured population provides a molecular basis for body color breeding of C. semilaevis in the future.