Genetic differentiations and phylogeographical patterns are controlled by the interplay between spatial isolation and gene flow. To assess the extent of gene flow across an oceanic barrier, we explored the effect of the separation of the peninsula of Baja California on the evolution of mainland and peninsular populations of the long-lived columnar cactusStenocereus thurberi. We analyzed twelve populations throughout the OPC distribution range to assess genetic diversity and structure using chloroplast DNA sequences. Genetic diversity was higher (Hd= 0.81), and genetic structure was lower (GST= 0.143) in mainland populations vs peninsular populations (Hd= 0.71,GST= 0.358 respectively). Genetic diversity was negatively associated with elevation but positively with rainfall. Two mainland and one peninsular ancestral haplotypes were reconstructed. Peninsular populations were as isolated among them as with mainland populations. Peninsular haplotypes formed a group with one mainland coastal population, and populations across the gulf shared common haplotypes giving support to regular gene flow across the Gulf. Gene flow is likely mediated by bats, the main pollinators and seed dispersers. Niche modeling suggests that during the Last Glacial Maximum (c. 130 ka), OPC populations shrank to southern locations. Currently,Stenocereus thurberipopulations are expanding, and the species is under population divergence despite ongoing gene flow. Ancestral populations are located on the mainland and although vicariant peninsular populations cannot be ruled out, they are likely the result of gene flow across the seemingly formidable barrier of the Gulf of California. Still, unique haplotypes occur in the peninsula and the mainland, and peninsular populations are more structured than those on the mainland.