Delineating population boundaries in anthropogenic landscape is of critical importance for domains of biology that are concerned with the ecology, evolution and conservation of species. This remains particularly difficult for species where there is no obvious demarcation of geographical populations and dispersal patterns are poorly known. This is often the case in amphibian species that reproduce in aquatic habitats but live otherwise in terrestrial habitats. Sampling of such species usually occurs in the aquatic habitat (i.e., breeding sites) but these may represent neither genetically nor demographically distinct populations. Here, we analyzed the genetic structure of a stream-breeding species, the fire salamander (Salamandra salamandra) along a rural-to-urban land use gradient. We used genetic data and Bayesian clustering approaches, which rely on genetic information without assuming predefined populations, to delineate distinctive genetic units along this gradient, and compare genetic diversity between rural and urban areas. The different analytical approaches used partitioned our dataset in slightly different but highly congruent clusters, that included localities which were up to 19 km apart from each other. Genetic breaks occurred at unexpected places while several landscape features reported to act as barriers on gene flow for amphibians did not lead to genetic breaks. Our results emphasize the difficulty to delineate management units in open systems, especially for long-lived species, for which the timespan between the establishment of a barrier and its translation into clear genetic breaks will take longer than in short-lived species.