DNA barcoding based on partial Cytochrome Oxidase subunit I (COI) gene in the mitochondrial has widely used in species identification and biodiversity studies. COI gene application is expected to obtain genetic characteristic, genetic variations and phylogeny of giant featherback. The aim of this research was to analyze genetic diversity of giant featherback Chitala lopis in Lampung and Kalimantan. To analyse genetic distance, Kimura two parameter (K2P) model was performed where to determine nucleotide variation & polymorphism and also reconstructed of phylogenetic tree was used MEGA 7.0 software. Total nine individuals were obtained from three populations, i.e. Lampung, West Kalimantan and South Kalimantan. The results showed that giant featherback has 689 bp conserve, 18 bp variation, 13 bp parsimony-informative, and 2 bp singleton sites from 707 bp COI partial gene. The average within-species, in-group, and out-group based on K2P distances were 1.24%, 1.43% & 1.58% (AP008922.1; KM213054.1), and 13.00% respectively. The Single Nucleotide Polymorphism (SNP) was obtaining from 13 SNP sites. West Kalimantan samples have two SNP (471 and 528 site). The South Kalimantan samples showed more specific nucleotides with nine SNP (120, 129, 144, 201, 306, 324, 474, 615 and 644). Based on genetic distance, the biggest difference was in the South Kalimantan sample (1.58%) compared with Lampung and West Kalimantan. The results of the K2P neighbour-joining phylogenetic tree reconstruction show that the South Kalimantan samples are in a different group. The West Kalimantan sample shows that it is closely related to the Lampung.