The present study investigated the genetical and morphological differentiation of Cynoglossidae family in Boushehr (Kangan, Boushehr, Deilam) and Khoozestan (Abadan) province. Morphometric, meristic and molecular characterization were calculated for three species of Cynoglossidae. Difference in body shape between species was conducted using traditional morphometric and geometric morphometric techniques, digital imagery and define different landmarks on two- dimensional images. cytochrome oxidase subunit I gene (COI) were used for studying genetically differentiated and phylogenetic relationship of Cynoglossidae family. COI sequence obtained in this study were phylogenetically compared to other Cynoglossidae sequences obtained from gene bank. morphometric data were analysed by multivariate principle component analysis (PCA), canonical variate analysis (CVA) and discriminant function analysis (DFA). The results show a significant shape difference between stations and the maximum difference related to Kangan and Abadan samples and minimum difference were between Boshehr and Deilam stations. Haplotype diversity was calculated 0/727 for Cynoglossus arel, Cynoglossus bilineatus and Cynoglossus dispar 0/667. Phylogenetic trees drived from COI gene sequence using Neighbor joining and Unweighted pair group methods showed significant differentiation in studing samples. In the present study, the average GC in Cynoglossus arel, Cynoglossus bilineatus and Cynoglossus dispar were 41/44, 41/13 and 41/48 respectively. The genetic distance between samples was calculated between 0/002-0/005, which indicates that the COI gene is an effective marker for identification and barcoding of the Cynoglossidae species . Overall, the results of this study showed geometric morphometric method is more efficient than traditional method. Our results can be used for species identification, ecologiucal managments and larval or fisheries products identification.