The mitochondrial Cytochrome C Oxidase 1 (CO1) gene is used as a standardized, authenticated and reliable molecular marker for a global species-level bio-identification system. The present study was conducted to analyze the DNA barcodes for accurate species identification from query samples. Complete COI gene (1551 bp) was amplified using PCR and sequenced from 26 fish samples collected from river Neelum and Jhelum Azad Kashmir. Out of 1551 sites of COI gene, 1490 (96.06%) sites were constant and 61 (3.93%) were variable characters in which 54 were parsimony informative, and 7 variable characters were singleton. The query sequences were tested against the public databases BOLD and Genbank. The overall mean genetic distance was 0.008, in which mean intraspecific nucleotide distance was 0.205% and relatively high interspecific distance (1.837%) was observed between present study and references sequences. Phylogenetic trees were also constructed to characterize the phylogenetic relationships of these species that showed the 99% to 100% sequence similarity with their corresponding species in the databanks. For the identification of fishes from different geographic origins, the open access reference libraries of DNA barcodes were used. High precision identification of species from DNA samples has major utility in different fields of fisheries conservation programs to reduce the fish vulnerability. Schizothoracinae commonly called snow trouts inhabit throughout the rivers of Azad Jammu and Kashmir. In the present study, four species of Schizothorax have been identified by using the molecular markers, while three species were already reported on the basis of their morphological characters (Schizothorax esocinus, Schizothorax plagiostomus and Schizothorax progastus) and one new species (Schizothorax niger) was identified first time in AJK. A contradicting relationship was observed among these species. To resolve the evolutionary relationships among these species, we sequenced mitochondrial fragments, including 16S rRNA, Cytb and D-loop region of mitochondrial DNA. The average nucleotide length of complete 16S rRNA in 45 samples of 4 Schizothorax species was ranged from 1527 bp to 1552 bp per specimen. Five haplotypes (h) were observed in these sequences, with haplotype diversity (Hd) 0.5323±0.080. Out of 1552 sites, 1547 (99.67%) sites were found to be conserved (monomorphic) and five are polymorphic. Out of five polymorphic sites, four are parsimony informative while one is singleton. 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. The Displacement loop was very mutable and showed the maximum length variations among species. The conserved sequence blocks were found in D-loop region of fish as compared to other vertebrate. Present findings indicates the presence of four conserved sequence blocks (CSB), four TAS motifs and 15 bp pyrimidine block in D-loop region. Total length of D-loop region varies from 763 to 777 bp in all the four Schizothorax species. The D-loop was found rich in AT contents and 5´ end of D-loop region was more conserved as compare to 3´ end. Out of 777 total characters, 760 (97.8%) characters were constant, all 17 (2.18%) variable characters were parsimony informative. The estimated transition/transversion bias (R) was 3.76. The sequence analysis of Cytb showed that out of 1148 sites, 1069 (93.11%) sites were conserved (monomorphic) and 79 (6.88%) were variable without any insertion or deletion. All these variable sites are parsimony informative. The rate of transition/transversion (R) was also higher in first codon position (R) is 53.81, which deviate from the neutral evolution (R= 0.5). A total of 9 haplotypes (h) were identified in Cytb gene of four Schizothorax species with average haplotype diversity (0.8420±0.038) and nucleotide diversity (0.01204±0.0051). Values obtained from different tests: Tajima’s D= -1.01919 (P > 0.10); Fu and Li’s D* = 1.96198 (P < 0.02); and Fu and Li’s F* = 1.09825 (P > 0.10). The negative values of Tajima’s D test shows the bottle neck effect whereas values of Li and Fu’S D* and Li and Fu’s F* test show excess of external mutations. The phylogenetic relationship analyzed by maximum likelihood and neighborjoining generated almost identical results suggesting that S. plagiostomus, S. esocinus and S. progastus were more closely related to each other than the S. niger, which was also confirmed by the genetic distance data but they have shared the common ancestor. A new species, S. niger inhabiting cold streams and rivers is distributed in the inland waters of occupied Kashmir but in present study S. niger was first time collected and identified from river Jhelum near Muzaffarabad city. The sequences of these genes were compared with other cyprinids fishes downloaded from NCBI along with one outgroup (Barbus barbus) as a root of tree. The results indicate that 16S rRNA, D-loop and cytochrome b genes are useful in analyzing genetic variation as well as in unravelling phylogenetic relationship in the subfamily Schizothoracinae. Current study gives a knowledge of molecular phylogeny of the species and enhances our comprehension of historical and taxonomic connections got from morphological and ecological studies. This is the very first study reported from a reservoir of cold water bodies of Azad Kashmir which have a great potential for conservation of cold water fish species.