Coryphoblennius galerita is a small intertidal fish with a wide distribution and limited dispersal ability, occurring in the northeastern Atlantic and Mediterranean. In this study, we examined Atlantic and Mediterranean populations of C. galerita to assess levels of genetic divergence across populations and to elucidate historical and contemporary factors underlying the distribution of the genetic variability. We analyse three mitochondrial and one nuclear marker and 18 morphological measurements. The combined dataset clearly supports the existence of two groups of C. galerita: one in the Mediterranean and another in the northeastern Atlantic. The latter group is subdivided in two subgroups: Azores and the remaining northeastern Atlantic locations. Divergence between the Atlantic and the Mediterranean can be the result of historical isolation between the populations of the two basins during the Pleistocene glaciations. Present-day barriers such as the Gibraltar Strait or the ‘Almeria-Oran jet’ are also suggested as responsible for this isolation. Our results show no signs of local extinctions during the Pleistocene glaciations, namely at the Azores, and contrast with the biogeographical pattern that has been observed for Atlantic–Mediterranean warm-water species, in which two groups of populations exist, one including the Mediterranean and the Atlantic coast of western Europe, and another encompassing the western tropical coast of Africa and the Atlantic islands of the Azores, Madeira and Canaries. Species like C. galerita that tolerate cooler waters, may have persisted during the Pleistocene glaciations in moderately affected locations, thus being able to accumulate genetic differences in the more isolated locations such as the Azores and the Mediterranean. This study is one of the first to combine morphological and molecular markers (mitochondrial and nuclear) with variable rates of molecular evolution to the study of the relationships of the Atlantic and Mediterranean populations of a cool-water species.