Knowledge of spatial patterns of genetic differentiation between populations is key to understanding processes in evolutionary history of biological species. Caulerpa is a genus of marine green algae, which has attracted much public attention, mainly because of the impacts of invasive species in the Mediterranean. However, very little is known about the ecological and evolutionary history of the Mediterranean native Caulerpa prolifera, a species which is currently found at sites distributed worldwide. C. prolifera provides a good model to explore the patterns of genetic diversity at different scales across the Mediterranean and Atlantic area. This study aims to investigate the biogeographical patterns of diversity and differentiation of C. prolifera in the Mediterranean, with special focus on the Mediterranean/Atlantic transition zone. We used two nuclear (ITS rDNA and the hypervariable microsatellite locus CaPr_J2) and one chloroplast (tufA) DNA markers on samples of C. prolifera from its entire range. Analyses of 51 sequences of the cpDNA tufA of C. prolifera, 87 ITS2 sequences and genotypes of 788 ramets of C. prolifera for the locus CaPr_J2 revealed three different biogeographical areas: West Atlantic, East Atlantic and a larger area representing the Mediterranean, the Mediterranean/Atlantic transition zone and a Pacific site (Bali). It was found out that the Mediterranean/Atlantic transition zone is a biogeographical boundary for C. prolifera. A lack of connectivity was revealed between Atlantic and Mediterranean types, and identical sequences found in the Mediterranean and Indo-Pacific suggest either recent gene flow along the Red Sea connection or a possible ancient Indo-Pacific origin.​