According to the existing mitochondrial gene sequence of Acrossocheilus parallens, primers were designed, and by using the method of primer walking and PCR amplification, mitochondrial genome sequence of A. kreyenbergii was obtained, and its structure was analyzed. The results showed that the complete mitochondrial genome of A. kreyenbergii was a circular genome, 16 596 bp in length, including 13 protein coding genes, 22 tRNA genes, 2 rRNA genes and two non-coding regions (D-loop and origin of light strand replication, OL). The base composition showed two features, the negative bias for G and AT bias. All protein-coding genes started with ATG as an initiation codon except COⅠ, which used GTG. Two complete termination codons were TAA (38.46%) and TAG (7.69%), and two incomplete stop codons were TA (15.38%) and T (38.64%). A control region of 941 bp in length between tRNAPro and tRNAPhe, a short tandem repeat TA, was found in the 811﹣837 bp intervals. From the information contained in the protein coding gene, the mutation site and the mutation rate of view, ND5, ND4, COⅠ and ND2 were the most suitable molecular markers for interspecific phylogenetic analysis of Acrossocheilus. The phylogenetic tree constructed by Bayesian method for 13 protein coding genes showed that 23 species of Acrossocheilus and Onychostoma were not clustered into monophyletic groups, and could not be clearly distinguished from each other.