Aim To elucidate the historical phylogeography of the dusky pipefish (Syngnathus floridae) in the North American Atlantic and Gulf of Mexico ocean basins.

Location Southern Atlantic Ocean and northern Gulf of Mexico within the continental United States.

Methods A 394‐bp fragment of the mitochondrial cytochrome b gene and a 235‐bp fragment of the mitochondrial control region were analysed from individuals from 10 locations. Phylogenetic reconstruction, haplotype network, mismatch distributions and analysis of molecular variance were used to infer population structure between ocean basins and time from population expansion within ocean basins. Six microsatellite loci were also analysed to estimate population structure and gene flow among five populations using genetic distance methods (FST, Nei’s genetic distance), isolation by distance (Mantel’s test), coalescent‐based estimates of genetic diversity and migration patterns, Bayesian cluster analysis and bottleneck simulations.

Results Mitochondrial analyses revealed significant structuring between ocean basins in both cytochrome b (ΦST = 0.361, P <0.0001; ΦCT = 0.312, P <0.02) and control region (ΦST = 0.166, P <0.0001; ΦCT = 0.128, P <0.03) sequences. However, phylogenetic reconstructions failed to show reciprocal monophyly in populations between ocean basins. Microsatellite analyses revealed significant population substructuring between all locations sampled except for the two locations that were in closest proximity to each other (global FST value = 0.026). Bayesian analysis of microsatellite data also revealed significant population structuring between ocean basins. Coalescent‐based analyses of microsatellite data revealed low migration rates among all sites. Mismatch distribution analysis of mitochondrial loci supports a sudden population expansion in both ocean basins in the late Pleistocene, with the expansion of Atlantic populations occurring more recently.

Main conclusions Present‐day populations of S. floridae do not bear the mitochondrial DNA signature of the strong phylogenetic discontinuity between the Atlantic and Gulf coasts of North America commonly observed in other species. Rather, our results suggest that Atlantic and Gulf of Mexico populations of S. floridae are closely related but nevertheless exhibit local and regional population structure. We conclude that the present‐day phylogeographic pattern is the result of a recent population expansion into the Atlantic in the late Pleistocene, and that life‐history traits and ecology may play a pivotal role in shaping the realized geographical distribution pattern of this species.