This study investigated the genetic characteristics of mitochondrial COⅠ?(mitochondrial cytochrome c oxidase subunit Ⅰ) and 16S rRNA gene fragments and their applicability in species identification and phylogeny of sole fish Through sequencing and downloading available data from the GenBank, we obtained COⅠ?gene fragments from 65 individuals of 24 species in 14 genera and 16S rRNA gene fragments from 62 individuals of 23 species in 14 genera of Soleidae, respectively. Results of base composition analysis showed that the A+T content was higher than 50% except for 16S rRNA sequence of B. luteum (49.8%). The proportion of variation base of COⅠ?(about 45.5%) was a little higher than that of 16S rRNA (about 44.3%). Means of intraspecific and interspecific K2P genetic distances were respectively 0.33% and 19.91% for COⅠ sequences, and 0.26% and 7.19% for 16S rRNA sequences. Genetic differences of COⅠ?sequences between congeners were, on average, about 60 times (minimum 19 times) higher than the differences within species, which met Hebert’s species identification standard (more than 10 times the difference between intraspecies and interspecies). Genetic differences of 16S rRNA sequences were 28 times between intraspecies and interspecies (minimum five times). Therefore, COⅠ and 16S rRNA sequences were both applicable for species identification in sole fish. Moreover, there were overlaps in genetic distance distribution of the two gene fragments between different taxa. The overlap region in COⅠ-based genetic distance distribution existed in higher taxa (interspecies, genus, family), while that in 16S rRNA sequence existed in lower taxa (intraspecies, interspecies, genus) 16S rRNA. As a result, it is advisable to select appropriate molecular markers according to different classified levels when applying the two gene fragments for studying systematic evolution.