Retama raetam is a stem-assimilating, C3, evergreen, medicinal plant species, desert legume common to arid ecosystems around the Mediterranean basin. This study addresses the genetic diversity and relationship among and within three populations collected from different habitats in southern Tunisia by Random Amplified Polymorphic DNA (RAPD). Estimates of the percentage of polymorphic bands, Shannon's diversity information index and Nei's gene diversity index were determined. Results showed that population from the Island Djerba has the lowest Nei's gene diversity; this also was for Shannon diversity index. An analysis of molecular variance indicated that the majority of variation existed within populations (68%) and that there was significant differentiation among populations (phiPT = 0.316, p < 0.001). Genetic distance (phiPT based values) between pairwise populations ranged from 0.098 to 0.505 and the differentiation between pair-wise populations was significant when individual pairs of populations were compared. Based on the coefficient of gene differentiation (Gst), gene flow (Nm) was estimated and was found to vary from 0.490 to 4.609 between pair-wise populations and 1.42 among populations. The results of UPGMA cluster analysis and PCoA analysis indicated that most variation occurred within populations and that genetic differentiation had happened between populations. These findings are important for a better understanding of the adaptive strategy of R. raetam in southern Tunisia and will be useful for conservation managers to work out an effective strategy to protect this important species.