Edge populations can harbor unique genetic diversity shaped by historical isolation and play a key role in species' resilience and range expansion under ongoing climate warming. The Aesculapian snake (Zamenis longissimus) reaches the northern limit of its range in Central Europe, where isolated populations may provide key insights into the species' evolutionary potential and conservation priorities. In Bohemia (the western Czech Republic), only one reproducing population, in the vicinity of Stráž nad Ohří (SO), had previously been confirmed north of the species' continuous distribution. Here, we report two additional reproducing populations recently discovered through long-term monitoring: one at the Želinský meander (ZM) and another in Central Bohemia (CB). The ZM population is autochthonous, viable, and genetically remarkable, harboring two divergent mitochondrial haplotypes: the widespread Eastern phylogeographical clade haplotype E1 and a novel haplotype, W10, belonging to the Western clade. This represents the first confirmed record of a Western clade haplotype in the Czech Republic, and only the second known locality within the species' entire range where both clades coexist. In contrast, the CB population-founded by human-mediated translocation from SO-is expanding dynamically and is represented solely by E1, the only haplotype previously recorded in the country. Our study highlights the importance of incorporating genetic data into conservation planning and understanding species' evolutionary potential. The mitochondrial diversity uncovered at the ZM exemplifies how historical processes, isolation, and lineage mixing shape contemporary genetic structure. Preserving such populations, which retain unique evolutionary diversity, will be critical for maintaining the resilience of Z. longissimus in Central Europe. More broadly, disjunct northern populations may serve as reservoirs of genetic diversity, enhancing adaptive potential and supporting future range expansion under climate change. Recognizing and conserving this diversity is essential not only for local persistence but also for species-level resilience in a rapidly changing environment.

• Keywords
• autochthonous population, climate change, coxI, cytb, genetic admixture, introduction, isolated populations, new haplotypes, rat snakes,
• Publication type
• Journal Article MeSH