Habitat destruction and fragmentation caused by highways can negatively affect animal populations, but a better understanding of the effects of highways on population genetic structure is still needed to improve conservation plans in urbanized landscapes. We investigated the degree of genetic variability and differentiation within and among seven Rana dalmatina populations located far from highly trafficked roads (non-fragmented populations) and four populations sampled on both sides of a major highway (fragmented populations). The degree of population subdivision was significantly higher among fragmented (F_ST = 0.238) as compared to non-fragmented populations (F_ST = 0.022). Furthermore, in the four fragmented populations, significantly lower allelic richness as compared to non-fragmented populations was observed. Together with potential high levels of road mortality leading to smaller population size, these results suggest that separation by highways not only has reduced the genetic diversity and polymorphism in local populations over two decades, but also has resulted in a higher degree of population differentiation, most likely due to genetic drift.