Abstract. Acacia longifolia is an aggressive invader worldwide. This species was brought to Portugal by the forestry services and is now found throughout the country with very strong ecological and social impacts. Although several ecological and physiological studies have been carried out, molecular studies in this species are sparse. Particularly, genetic variability evaluation in invasive ranges clearly deserves more attention. The aim of this study was to evaluate the genetic diversity and the genetic differentiation among populations of the alien invasive A. longifolia under different Mediterranean conditions. For that we studied three sandy dune populations along the Portuguese coast: Osso da Baleia (mesomediterranean), Pinheiro da Cruz and Vila Nova de Milfontes (termomediterranean). All sampled acacia plants were located underneath a pine forest, with the exception of the latter case study, where we also sampled in a diversified habitat conditions associated with the margins of agriculture fields. According to the historical records, it was also possible to compare acacia samples from original plantations and recently invaded areas. We have used two molecular markers – inter-simple sequence repeats (ISSR) and microsatellites (SSR). ISSR analysis showed that A. longifolia populations at all sampled locations have similar levels of diversity and a relatively low differentiation (ΦPT=0.135). However, samples tended to cluster according to the regional (macro-scale) collection site. Microsatellites confirmed this low differentiation pattern. Our results indicate that differences in phenology and reproductive success observed during a previous study cannot be accounted for by genetic diversity and differentiation alone. Furthermore, our results taken together with historical information of A. longifolia suggest a single original introduction and a subsequent acclimation process.