The Strait of Gibraltar (SG) is reputed for being both a bridge and a geographic barrier to biological exchanges between Europe and Africa. Major genetic breaks associated with this strait have been identified in various taxa, but it is unknown whether these disjunctions have been produced simultaneously or by independent biogeographic processes. Here, the genetic structure of five conifers distributed on both sides of the SG was investigated using mitochondrial (nad1 b/c, nad5-1, nad5-4 and nad7-1) and chloroplast (Pt1254, Pt15169, Pt30204, Pt36480, Pt71936 and Pt87268) DNA markers. The distribution of genetic variation was partially congruent between types of markers within the same species. Across taxa, there was a significant overlapping between the SG and the genetic breaks detected, especially for the four Tertiary species surveyed (Abies pinsapo complex, Pinus nigra, Pinus pinaster and Taxus baccata). For most of these taxa, the divergence of populations across the SG could date back to long before the Pleistocene glaciations. However, their strongly different cpDNA GST and RST values point out that they have had dissimilar population histories, which might include contrasting amounts of pollen-driven gene flow since their initial establishment in the region. The fifth species, Pinus halepensis, was genetically depauperated and homogenous on both sides of the SG. A further analysis of nuclear DNA sequences with coalescent-based isolation with migration models suggests a Pleistocene divergence of P. halepensis populations across the SG, which is in sharp contrast with the pre-Pleistocene divergence dates obtained for P. pinaster. Altogether, these results indicate that the genetic breaks observed across this putative biogeographical barrier have been produced by independent evolutionary processes related to the biological history of each individual species instead of a common vicariant phenomenon.