Urban grasslands provide numerous ecosystem services, and their maintenance should be based on naturally regenerating plant populations. However, the urban environment is challenging for preserving viable populations, mostly because of their high fragmentation and small size, which can lead to genetic drift. We examined red clover (Trifolium pratense) in a medium-size city in Central Europe to test the cityscape effect on within- and among-population genetic diversity. We used eight inter-simple sequence repeat markers to examine the genetic structure of 16 populations, each represented by eight individuals. The isolation by resistance was analysed using a least cost patch approach, focusing on gene flow via pollinators. We found great variation amongT. pratensepopulations, with no discernible geographic pattern in genetic diversity. We linked the diversity to the long history of the city and high stochasticity of land use changes that occurred with city development. In particular, we did not find that the Odra River (ca. 100 m wide) was a strong barrier to gene transfer. However, notable isolation was present due to resistance and distance, indicating that the populations are threatened by genetic drift. Therefore, gene movement between populations should be increased by appropriate management of urban green areas. We also found that small urban grassland (UG) patches with small populations can still hold rare alleles which significantly contribute to the overall genetic variation ofT. pratensein the city.