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The black sea cucumber Holothuria leucospilota is a commercially exploited species in the Western and Central Pacific region including Australia and Vietnam, where it has been overfished. There is interest to develop captive breeding, sea ranching and restocking programs for H. leucospilota. However, the wide distribution of this species could lead to genetic structure among site, which needs to be considered for responsible genetic management for example, restocking. This is the first study to investigate population genetics in H. leucospilota, between Australia and Vietnam, using single nucleotide polymorphisms (SNPs). The results indicated that there was high gene flow (Nm > 4) and genetic similarities among three Vietnamese populations, which inferred a high panmictic stock structure in Vietnam. Less gene flow (Nm > 1) was indicated between Vietnam and Mooloolaba (Eastern Australia) populations, and there was a significant genetic differentiation among any Vietnamese populations and Mooloolaba population (FST from 0.034 to 0.043; P < 0.05). Surprisingly, there was a major genetic difference (FST > 0.2) and a restricted gene flow (Nm <1) between Darwin (Northern Australia) population and other sea cucumber populations studied (Vietnam and Eastern Australia), which revealed a genetic break/ divergence in Darwin. Based on these findings, we can propose that the Vietnamese sea cucumber stocks be considered as a single genetic stock with concordant implications for genetic management. We consider whether the genetic differences in the Darwin stock arise from bottlenecking and are not reflective of adaptive differences. In summary, the results from this study provide basic genetic information on H. leucospilota populations, that would help to design restocking management plans, genetic conservation initiatives and sea ranching programs for H. leucospilota.