Molecular phylogeny of Schizothoracinae containing four species distributed in the north and north-east Himalayas was investigated based on the complete mitochondrial 16S rRNA gene sequences. The average nucleotide length of 16S rRNA in 45 samples ranged from 1527 to 1552 bps. Five haplotypes (h) were observed, with haplotype diversity (Hd) 0.5323±0.080. Among 5 haplotypes, 2, 2 and 1 haplotype were detected in S. plagiostomus, S. niger and S. esocinus, respectively. Haplotype diversity was lowest and same in S. esocinus, S. niger and S. progastus (0.0001) and highest in S. plagiostomus (0.5436). Average number of nucleotide differences of 4 species (Kt) was 0.737 and total nucleotide diversity (Pi) was equal to 0.0004 ± 0.0001. Out of 1552 sites, 1547 sites were found to be conserved and five were polymorphic, in which four were parsimony informative while, one was singleton. The rate of transition/transversion (R) was 3.131, which deviate from neutral evolution where, R= 0.5. The negative values of Tajima’s D and Fu and Li’s F* indicated that the genetic variations in 16S rRNA between species were not neutral which reflect the excess of external mutations. Results indicate that 16S rRNA gene is unable to fully resolve the inter-relationship of Schizothoracinae species.