The widespread farming of catfish (genus Clarias) hybrids between bighead catfish (C. macrocephalus, Cm) and the introduced North African Catfish (C. gariepinus, Cg) in the Mekong Delta, where flooding is common, has raised concerns of introgression of Cg into the native catfish due to backcrossing by escaped hybrids with wild Cm. This study employed novel PCR-RFLP analyses of a mitochondrial (Cytochrome c oxidase subunit I, COI) and two nuclear (Rhodopsin and Tropomyosin) markers and six microsatellite loci to differentiate hybrid individuals from their parental species, and evaluate whether introgression has occurred in wild and cultured Cm populations in the region. Results of marker screening showed that sizes and frequencies of microsatellite alleles differed between the two parental species. Four loci exhibited fixed species-specific differences in allele frequency (monomorphic in Cg and highly polymorphic in Cm). Levels of sequence variation varied at the three genes examined, ranging from 3 to 8% (sequence length 652 bp for COI, 802 bp for Rhodopsin and 925 bp for Tropomyosin). Based on variable sites identified that were not shared between the two species, 3 restriction enzymes (SpeI, XcmI and PflMI) were selected to digest PCR products at species-specific sites within the COI, Rhodopsin and Tropomyosin genes, respectively. Results of PCR-RFLP analyses confirmed that Cm is the maternal lineage of cultured hybrids, while nuclear genes (Rhodopsin and Tropomyosin) and microsatellite loci revealed that hybrids possessed admixed multi-locus genotypes relative to the two parental species. Analyses of six microsatellite loci and PCR-RFLP of two nuclear genes in 473 individuals collected from 11 wild and three cultured populations revealed one F1 hybrid in the wild but no evidence of widespread introgression into native bighead catfish. These results differ from findings in Thailand where intensive catfish culture and deliberate species hybridization is practiced.