The wild stock of Indian major carps is declining in major rivers of India due to pollution, overexploitation, and inbreeding depression. In the present study, the comparative population structure, genetic diversity, and historical demographics of IMCs, Labeo rohita, Labeo catla, and Cirrhinus mrigala were characterized by analyzing partial 307bp sequences of Cytochrome b gene of 357 individuals collected from seven geographically isolated sites from Indian river basins and two culture zones. The DNA sequences alignment among three species revealed comparative divergence and evolution in these heterogeneous species in terms of over transition to transversion ratio of 6.25. The study implicated the occurrence of 35 haplotypes with haplotype diversity of 0.7333 in L. rohita at Brahmaputra basin showing allelic richness, while no nucleotide diversity (π) 0.000 was observed in L. catla of two geographically isolated locations of Rivers Ganga and Narmada. The analysis of molecular variance (AMOVA) revealed the genetic diversity of IMCs is to be very low (9.40%) within the species, as compared to among the three species (80.76%). The Fst scores ranged between 0.000 and 0.996, indicating low to high genetic structure of IMCs. The Haplotype-1, Haplotype-13, and Haplotype-19 in L. rohita, L. catla, and C. mrigala were considered as ancestral haplotypes, respectively. The expansion factor was found to be 0.45000 and 0.90000 for L. rohita stocks at Farakka-Ganga and Allahabad-Ganga, respectively. The present study provides the first report on genealogical biogeography, evolutionary divergence, and expansion of three IMCs, which could help to formulate the strategies for conservation of fisheries biodiversity in the Indian riverine ecosystems.