Calcareous grasslands are among the most species-rich plant communities in Europe with a particularly high nature conservation value. During the past centuries their distribution has markedly decreased, at least partly due to urbanization. Thus we investigated the effects of urbanization on species diversity along a spatio-temporal urbanization gradient from traditionally managed grassland to areas affected by urban developments, which was situated in the plains northwest of Munich, Germany. Both a RLQ analysis linking species and environmental traits, and a redundancy analysis of the plant community features showed that soil disturbance, soil sealing and mean temperature explained most of the environmental variation along the gradient. The species in urban habitats showed increased insect pollination, earlier flowering and prolonged seed longevity. While urbanization favored short-lived species with dysochorous dispersal, the reference grasslands harbored more wind-pollinated perennials with effective vegetative spread and relatively large, short-lived seeds. Compared to the urban sites, traditionally used grasslands had a higher species diversity, more threatened species and a lower proportion of non-natives. We conclude that even under conservation management, urban habitats are not capable of maintaining the original biodiversity. However, we also found threatened species occurring exclusively in urban sites. Hence, urbanization decreased the area and diversity of traditional calcareous grasslands, but it also established niches for endangered species which are not adapted to the living conditions in calcareous grasslands.