The Nemacheilus subfusca (N. subfusca) is a species of the Noemacheilinae in the Cobitidae family, which has been few reported. The first complete mitochondrion of the N. subfusca is 16 585 bp in length, containing 13 protein-coding genes, two rRNA genes (12S and 16S rRNA), 22 tRNA genes and a control region (D-loop region). The longest protein-coding gene of this mitochondrion was ND5 (1839 bp), and the 22 tRNA genes were range from 66 (tRNACys ) to 76 (tRNALys ) in length. The 12S rRNA (957 bp) and 16S rRNA (1677 bp) were separated by TrnaVal , and the D-loop region located between tRNAPro and tRNAPhe genes. The phylogenetic indicates that the N. subfusca was independent from other species of distinct genus in the Noemacheilinae, which provided the valuable evidence on phylogenetic relationship of the N. subfusca at the molecular level.