The fish in the genus Schizothorax from the Cyprinidae family live in high-altitude Rivers andstreams, are threatened by various anthropogenic stressors. This study aims to characterize S. plagiostomus across Pakistan and throughout the world available on NCBI using the mitochondrial D-loop region, and in particular, to assess the degree of intra-specific pairwise distance among these sequences, as well as to establish their phylogenetic relationships. The percent overall nucleotide composition was 32.6% (A), 33.6% (T), 19.8% (C), and 14.0% (G), which infers that S. plagiostomus control regions is AT-rich (66.2%) and poor in G contents. The mean pair-wise intra-specific nucleotide diversity (Pi)of all the S. plagiostomus was 0.022. While, the inter-specific nucleotide diversity of all the Schizothorax species was 0.049. D-loop sequences for intra-specific variations revealed 765 sites were invariable and 10 were variable, 8 parsimony informative sites and only 2 were singletons. The overall transition/transversion ratio is R = 7.135. Three domains in S. plagiostomus were observed, namely, the termination associated sequence (TAS) domain, the central conserved sequence block (CSB) domain, and the conserved sequence block (CSB) domain. No substitution saturation was detected as an Iss value was significantly (𝑃< 0.001) lower than the Iss.c in all cases indicating the suitability of the data for phylogenetic analysis. This study signifies the importance of the control region for the genetic analysis of S. plagiostomus and also provides a hypothesis of their phylogenetic relationships.