Abstract The Cytochrome b gene from mitochondrial DNA encodes a protein that can be used as a genetic marker to identify species. This study aims to determine the bioinformatic characteristics of the gene encoding Cyt b isolated from various Mahseer species, Tor tambroides, Tor douronensis, Tor khudree, and Tor putitora. Mahseer Cyt b sequences can be used for detailed computational investigation of the properties of 3D protein models and phylogenetics. The data analysis technique uses nucleotides with a sequence length of 330 bp obtained from NCBI. The MEGA X program was used for phylogenetic analysis. 3D protein structure predictions were obtained through the Swiss model and protein homology program using PYMOL software. The results of phylogenetics show close kinship relationships with the closest genetic distance of 0.000, while the farthest distance is 0.068. The nucleotide composition contains Thymine (30.23%), Cytosine (25.81%), Arginine (28.05%), and Guanine (15.92%). 3D modeling of the Cyt b protein Mahseer referring to the species Neolissochilus hexagonolepis (McClelland, 1839). The 3D protein structure prediction was assessed via a Ramachandran plot of 99.07%. This study can be used as a reference to support sustainable conservation efforts by observing the populations and habitat conditions.
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