Catfish hybrids (Clarias macrocephalus x C. gariepinus) have been cultured popularly in Vietnam and have possibly escaped to the wild. Identifying hybrid individuals has become important in fisheries management and aquaculture, though hybrid identification based on morphology is highly uncertain. This study employed PCR-RFLP method using a mitochondrial (cytochrome c oxidase subunit I, COI) and a nuclear (rhodopsin, rho) markers to differentiate hybrids from their parental species. Two genes were sequenced in 12 samples of two species (6 of each species) and 3 samples of the culutred hybrid. Sequences of the two species were aligned to find species-specific restriction enzymes. Restriction enzymes of SpeI and XcmI were selected to digest at species-specific sites of COI and rho genes, respectively. Results confirmed that C. macrocephalus is maternal lineage of the cultured hybrid. Sequence chromatogram and fragments after XcmI digestion of rho gene of the hybrid reveal intermediate patterns between two parental species. Therefore, PCR-RFLP analysis of COI and rho genes is an effective and accurate method for identification of catfish hybrid individuals.