Puntius is an important genus of freshwater food and ornamental fish belonging to the family Cyprinidae. A total of 60 samples from twelve species of the genus Puntius were collected from eight sampling sites of eight Indian Rivers. Twelve species of Puntius (P. chola, P. sophore, P. filamentosus, P. fasciatus, P. vittatus, P. chelynoides, P. gonionotus, P. denisonii, P. ticto, P. gelius, P. conchonius and P. sarana) were investigated using 60 partial sequences of the mitochondrial cytochrome b (Cyt b, 1096 bp) gene to estimate genetic divergence and to establish the phylogenetic relationship. The average intraspecies diversity was estimated as 0.002, whereas the average interspecies diversity was estimated as 0.177. The sequence analysis of the Cyt b gene revealed four distinct groups, which are genetically distinct species and exhibited identical phylogenetic relationship. The present study validated the utility of the Cyt b gene in genetic diversity and phylogenetic studies.