Abstract: Collichthys lucidus and C.niveatus are commercially important fishery resources in China. Partial nucleotide sequences of mitochondrial DNA 16S rRNA and Cyt b gene fragments were sequenced and analyzed to study the relationship between C.lucidus and C.niveatus. Forty-four nucleotide substitutions were detected among 16S rRNA (526 bp) and Cyt b (379 bp) fragments. Nucleotide composition analysis indicated that the content of guanine (G) was relatively low in the two fragments, especially at the third codon position of Cyt b genes. Average genetic distances based on 16S rRNA and Cyt b between C.lucidus and C.niveatus were 0.012 and 0.111, respectively. The phylogenetic tree showedthat there was significant genetic divergence between C.lucidus and C.niveatus. According to the evolutionary rate of 2% perone million years, the estimated divergence time between C.lucidus and C.niveatus was 5.5 million years, which indicated an early Pliocene divergence for them.