Restriction fragment length polymorphism (RFLP) analysis of genomic DNA was employed to characterize species of Puntius sophore complex, a benthopelagic freshwater fish species. Samples of five species of Puntius viz, Puntius sophore, Puntius amphibius, Puntius chola, Puntius bimaculatus and Puntius dorsalis from the East-flowing Tamiraparani and Kuzhithurai and West-flowing Tamiraparani river basins (Western Ghats) were analyzed. The restriction enzyme employed (Hind III) generated species-specific DNA patterns for each of the five species. DNA exhibited considerable polymorphism among species. All samples showed relatively high values of diversity. The relation between species is discussed.