Striped snakehead (Channa striata) has been cultured for several decades in Viet Nam while only recently cultured in Cambodia. A cross-country comparison of genetic diversity in wild and cultured striped snakehead populations provided novel insights for improved genetic resource management and cultivation in Lower Mekong region. We collected striped snakehead samples from three wild and three cultured populations in the Vietnamese Mekong Delta for comparison to samples from eight wild populations in Cambodia (n = 5 and 3 from Tonle Sap Lake and Mekong River floodplain, respectively). Sequencing of cytochrome b (585 bp) and D-loop (874 bp) from 270 individuals yielded 28 and 128 haplotypes, respectively, resulting in 150 concatenated haplotypes. Mean genetic diversity indices of concatenated sequences were highest in wild Tonle Sap Lake populations (haplotype diversity Hd = 0.994 ± 0.004, nucleotide diversity pi = 0.0077 ± 0.0009), intermediate in wild Cambodian Mekong River populations (Hd = 0.925 ± 0.097, pi = 0.0076 ± 0.0005) and wild Vietnamese Mekong River populations (Hd = 0.832 ± 0.152, pi = 0.0061 ± 0.0018), and lowest in cultured Vietnamese populations (Hd = 0.451 ± 0.198, pi = 0.0021 ± 0.0002). The wild Tonle Sap Lake and cultured Vietnamese populations differed significantly in all genetic diversity indices (P < 0.05). The unique haplotypes and significant genetic divergence (P < 0.01) among striped snakehead populations from each habitat (e.g., lake or floodplain, wild or cultured) suggests habitat-specific genetic structure. Genetic differences among all wild populations were also positively correlated with hydrological distance in the range of 600 km (P < 0.01), suggesting isolation by distance. These findings have important implications for appropriate management of wild and cultured C. striata in Viet Nam and Cambodia.