The present study examined partial cytochrome b gene sequence of mitochondrial DNA for polymorphism and its suitability to determine the genetic differentiation in wild Labeo rohita. The 146 samples of L. rohita were collected from nine distant rivers; Satluj, Brahmaputra, Son, Chambal Mahanadi, Rapti, Chauka, Bhagirathi and Tons were analyzed. Sequencing of 307 bp of Cyto b gene revealed 35 haplotypes with haplotype diversity 0.751 and nucleotide diversity (π) 0.005. The within population variation accounted for 84.21% of total variation and 15.79% was found to among population. The total Fst value, 0.158 (P < 0.05) was found to be significant. The results concluded that the partial cyto b is polymorphic and can be a potential marker to determining genetic stock structure of L. rohita wild population.