The population structure of Labeo calbasu from 11 rivers belonging to the Indus, Ganges, Bhima, Mahanadi, and Godavari basins was investigated using allozyme marker systems. Seven out of 20 allozyme loci (35%) were polymorphic (P < 0.99). Both probability and score tests indicated significant deviation of genotype proportions from Hardy-Weinberg expectations at two loci, XDH* (Mahanadi, Bhima, and Godavari) and G6PDH* (Mahanadi). A pairwise genetic homogeneity test and F (ST) values indicated a low-to-moderate level (0.0515) of genetic structuring in the wild population of L. calbasu. AMOVA analysis also indicated moderate differentiation among the samples from different river basins. Analysis for genetic bottleneck was performed under the infinite allele model. The study revealed nine genetic stocks of L. calbasu from the natural population across Indian rivers. Evidence of genetic bottlenecks in some rivers was also revealed.