Clarias catfish hybrids (between C. macrocephalus, Cm, and C. gariepinus, Cg) 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 6 microsatellite loci and PCR-RFLP of a mitochondrial (cytochrome c oxidase subunit I, COI) and two nuclear (rhodopsin, rho and tropomyosin, trop) markersto differentiate hybrid individuals from their parental species. Size and frequencies of microsatellite alleles are different between the two parental species, in which 4 loci are monomorphic in Cg and highly polymorphic in Cm. Sequence similarities vary among three genes, ranging from 92 to  97% (sequence length 652 bp for COI, 802 bp for rho, and 952 bp for trop). Based on variable sites between species, 3 restriction enzymes of SpeI, XcmI, and PflMI were selected to digest at species-specific sites of COI, rho, and tropomyosin genes, respectively. Results of PCR-RFLP confirmed that C. macrocephalusismaternal lineage of the cultured hybrid. Sequence chromatogram and digested fragments of nuclear genes (rho and trop), and microsatellite loci of the hybrid revealintermediate patterns between two parental species. Six microsatellite loci and PCR-RFLP of three mitochondrial and nuclear genes used in the study have proved to be effective and accurate markers for identification of catfish hybrid individuals.