North Atlantic and South Atlantic swordfish (Xiphias gladius L.) are currently managed as two stocks separated at 5°N. While previous studies of genetic population structure using both mitochondrial and nuclear DNA confirm two genetically distinct stocks, sampling coverage has not been uniform or representative of all areas and estimates of admixture in areas of contact have not been provided. In this study, we examined: 1) the applicability of high-resolution melting analysis (HRMA) in population genetic studies of non-model organisms, 2) the use of nuclear markers in Atlantic swordfish and the methodology whereby nuclear gene variation can be quickly screened, identified, and genotyped using short-amplicon (SA) HRMA and unlabeled probe (UP) HRMA, and 3) the use of HRMA to characterize nuclear markers to study the genetic population structure of Atlantic swordfish using representative samples of the entire basin to provide an estimation of population admixture by means of Bayesian individual assignment. High resolution melting analysis (HRMA) is shown to be a highly sensitive, rapid, closed-tube genotyping method amenable to high throughput and, though until recently primarily confined to clinical studies, suitable for population studies in non-model species. Ten nuclear markers were genotyped primarily by SA- and UP-HRMA in North Atlantic (n=419), South Atlantic (n=296), and Mediterranean (n=59) swordfish. Comparisons of pairwise FST, AMOVA, PCoA, and Bayesian individual assignments were congruent with previous finding of three discrete populations with comparatively low levels of estimated gene flow for a marine organism (FST = 0.039-0.126). Population admixture was identified and estimated in the Northeast Atlantic and appeared to be asymmetrical, with swordfish from the South Atlantic found among North Atlantic localities but no North Atlantic migrants identified in the South Atlantic. The Mediterranean boundary currently at the Strait of Gibraltar is found to extend west into Atlantic waters to approximately 8°W. Similarly, the boundary between North and South Atlantic swordfish should be revised to a line that extends north from 0°N 45°W to 25°N 45°W and from that position, as a nearly horizontal line, eastwards to the African coast. Finally, I show that Bayesian individual assignment using the developed marker set can be used for mixed stock allocation in the Northeast Atlantic.