The nucleotide sequence of the complete mitochondrial DNA (mtDNA) genome was determined in Hippocampus trimaculatus. The complete mitogenome of H. trimaculatus was 16,529 bp with the GenBank Accession No. KJ956892, and it consisted of 13 protein-coding genes, 22 tRNA genes, 12S rRNA gene, 16S rRNA gene and 1 control region (D-loop). The heavy DNA strand (H-strand) carried most of the genes: 12 protein-coding genes, 2 rRNA and 14 tRNA. ND6 and eight tRNA genes were encoded on the L-strand. The lengths of 22 tRNA genes ranged from 66 to 76 bp, and all of them could fold into the typical cloverleaf-shaped secondary structure. The lengths of intergenic nucleotide are range from 1 to 14 bp with 7 overlaps inside. While four of the seven overlaps??ATP8 and ATP6, ATP6 and COIII, ND4L and ND4, ND5 and ND6?? are typical overlap sites of the teleost. The overall base composition of H. trimaculatus is 32.7% A, 29.3% T, 14.6% G and 23.4% C, with a slight A + T rich feature (62.0%). The gene order and composition of H. trimaculatus were similar to those of most other vertebrates. Two phylogenetic trees constructed by Neigbour-joining and Bayesian methods showed basically the same result. Seven species of genus Hippocampus clustered into a branch, which supported the existing classification of these seahorses.