To characterize the mitochondrial genome of the threespot wrasse Halichoeres trimaculatus (Perciformes, Labridae), we cloned and sequenced the complete mitochondrial DNA. The H. trimaculatus mitochondrial genome is 16,621 base pairs (bp) in length and consists of 13 protein-coding genes, 22 transfer RNA (tRNA) genes, two ribosomal RNA (rRNA) genes, and a control regions, as found in the mitochondrial genomes of other bony fishes. Most of the H. trimaculatus genes are encoded on the L-strand, whereas the nicotinamide adenine dinucleotide dehydrogenase (ND) 6 and eight tRNA (Gln, Ala, Asn, Cys, Tyr, Ser-UCN, Glu, and Pro) genes are encoded on the H-strand. Two reading-frame overlaps were found on the same strand: ATPase subunit (ATP) 8 and 6 overlapped by ten nucleotides, and ND4L and ND4 overlapped by seven nucleotides. The L-strand replication origin (31 bp) was located between tRNA-Asn and tRNA-Cys genes in the H. trimaculatus mitochondrial genome. The control region, which spans 861 bp, is located between tRNA-Pro and tRNA-Phe that shows consistency with its location as other bony fishes. Within this sequence, we identified several domain sequences such as a termination associated sequence and the conserved sequence blocks 1, 2, 3 as well as D box domains. These results provide basic information for comparative mitochondrial DNA in the genus Labridae along with further consideration on molecular phylogeny and biogeographic studies in the Halichoeres species.