Abstract: To analyze the characteristics of the sequences and to calculate the genetic distance within and between species, the 648 bp mitochondrial COⅠ gene was used as DNA barcode to analyze phylogenetic relationships of Botiinae fishes in 131 sequences from 19 species representing 7 genera of Botiinae. MEGA5.0 software was used in the current study. Phylogenetic analyses were carried out using neighbor-joining and Bayesian methods. Our result revealed that the nucleotide compositions were 24.4% A, 29.5% T, 18.0% G, and 28.1% C. The mean inter- and intra-specific genetic distances were 0.1480.008 and 0.0020.000, respectively. Individuals of each species were clustered into monophyletic groups based on the molecular phylogenetic tree, indicating that the mitochondria COⅠ gene is suitable for species discrimination. Moreover, the phylogenetic analyses supported monophyly of genera within Botiinae. Two clades were recovered within the subfamily Botiinae: one clade includes the sister-group of Leptobotia and Parabotia and the other contains Botia + Chromobotia and Sinibotia + (Syncrossus, Yasuhikotakia). This study indicated that the mitochondria COⅠ gene is a potentially effective marker for DNA barcoding and phylogenetic studies of Botiinae fishes.