The Cynoglossidae fish is an important resource of germplasm in marine fish.In this study,21 measurable characters of four groups of Cynoglossus fish including one cultured population of Half-smooth tongue sole Cynoglossus semilaevis(Günther,1873)(Group Cs),one wild population of Roule's tongue-sole Cynoglossus roulei(Wu,1932)(Group Cr)and two wild populations of Three-lined tongue sole Cynoglossus abbreviatus(Gray,1832)(Group Cas,which was divided into Group Ca1 and Group Ca2)were compared using One-way ANOVA and Student-Newman-Keuls multiple comparison method.The results showed that there is no significant difference between Group Ca1 and Group Ca2,while significant difference existed between any other two groups.Furthermore,the gene fragments of cytochrome oxidation enzyme I subunits(COⅠ)and the cytochrome b(Cyt b)in mitochondria(mtDNA)were cloned and analyzed from the four tongue sole groups respectively.The intraspecific and interspecific genetic distances of the four tongue sole groups were further analyzed based on the COⅠ and Cyt b genes.Both morphological and phylogenetic analysis proved that Half-smooth tongue sole and Roule's tongue sole had a closer relationship,while it was far from Three-lined tongue sole.N-J phylogenetic evolutionary analysis indicated that COⅠ and Cyt b genes can be used as DNA barcoding in germplasm identification of these three tongue sole species.This study provided a clear genetic background for cross breeding,selective breeding and system development research for the three species of tongue sole fish.