Abstract Stellaria holostea is a clonal herb highly confined to well-established deciduous forests. This study examined whether its genetic diversity and spatial genetic organization in urban forest islands is similar to the values from well-established populations outside urban landscape. We studied four populations in Warsaw and two populations from well preserved forests outside the city. Genetic diversity was greater in populations from well-preserved forests than from forests heavily exploited in the past. High clonal diversity indices indicate that the studied populations did not lose the ability to reproduce sexually. The small populations in urban forests differed from the remaining sites in spatial organization. High overall FST (0.24) and the lack of correlation between genetic and geographic distance between the studied populations indicate limited gene flow. Urban forests may be of great value for conservation of S. holostea and other ancient forest species as they may still harbor substantial genetic variability despite their isolation.