The genus Butis comprises fishes with relatively small body size and wide salinity tolerance allowing them to adapt to various environments. This research aims to analyze their phylogenetic relationships through the mitochondrial cytochrome b gene (Cyt b) sequencing. The results revealed that the population of Butis species had nine haplotypes with a haplotype diversity index of 0.67–1.00. The Cyt b gene sequences were rich in %C (30–31%) and low in %G (14–16 %). %AT was always higher than %GC while the AT/GC ratio was constant between Cyt b sequences. The intraspecific genetic distances were 0.001–0.004; meanwhile, the interspecific genetic distances were 0.003–0.203. All B. koilomatodon were in clade I of the phylogeny with a bootstrap value of 69%. Butis humeralis and B. butis were not grouped by species as B. koilomatodon but interspersed in the sub-clades of clade II with bootstrap values as high as 100%. Simultaneous use of mitochondrial DNA and nuclear DNA barcoding is recommended to conduct more precise identification of B. humeralis and B. butis in future research due to their great similarity in morphology and Cyt b gene sequences.