The spiny lobster Panulirus argus is one of the most important fisheries resources in the western tropical Atlantic Ocean. This valuable species is under heavy exploitation and there is an urgent need for information on its population structure and genetic variability in order to assist the design of conservation and fishery management strategies. Understanding of the population dynamics is critical in order to accurately describe management units and prevent elimination of stocks. In the present study genetic markers are introduced for the first time in this species and the population genetics of this species is studied. Nineteen microsatellite markers (mostly tetranucleotides repeats) have been isolated and characterised in P. argus. The hypervariable domain in the control region (CR) of the mitochondrial DNA was also used as a marker to study population subdivision. Both proved to be very useful for delineating population structure and geographic variation of P. argus throughout its distribution. Samples were obtained from across the P. argus distribution (southeastern Brazil to Florida). Analyses with both markers revealed a very high level of genetic divergence, warranting higher taxonomic separation between populations sampled in the Caribbean and those off the coast of Brazil, thus indicating strong genetic discontinuity, possibly influenced by the Amazon River and Orinoco River massive discharge between these two macrogeographical regions. In contrast, populations within Caribbean and Brazilian waters did not display significant genetic differentiation, with genetic variability randomly distributed among populations suggesting panmixia within these geographical regions.