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Crocidura suaveolens is a rare, habitat-specialist species in the Gulf of Cadiz (southwestern Iberia), the southwesternmost limit of its distributional range. In this region, it is present only in six tidal marshes distributed in four isolated areas as a result of competitive exclusion by C. russula. These rear-edge populations of C. suaveolens could have a high conservation value because they are predicted to contain high and unique genetic diversity, given their isolation in climatic refugia through Quaternary climatic oscillation. Here, we performed a genetic characterization of these populations using 10 microsatellite loci as a first assessment of their conservation status and to guide their conservation and management. A total of six genetic clusters of C. suaveolens were identified, corresponding to the six marshes sampled. Levels of differentiation among them were correlated with geographic distance, except for the high differentiation of one site (Estero Domingo Rubio [EDR]) despite its close proximity to two other sites (Tinto and Odiel), probably as a consequence of recent anthropic isolation. However, moderate levels of local and high levels of regional genetic diversity were observed, making the Gulf of Cadiz a region with a great evolutionary potential to face future threats. The four isolated tidal marsh areas occupied by the species should be treated as management units.