The present study applied a next-generation sequencing approach to determine the entire mitochondrial genome sequence of Melanotaenia fasinensis, an endemic species confined in the karst landscape belonging to the family of Melanotaeniidae. The mitochondrial genome is comprised of 13 protein-coding genes. The utilization of a phylogenetic tree that relies on 13 protein-coding genes (PCGs) offers significant molecular information that can be used for subsequent analysis of the phylogeographic and evolutionary aspects of Melanotaeniidae.