Calcareous grasslands are biodiversity hotspots in Western Europe. In Belgium, a number of phytosociological surveys have been realized in these habitats, but none covers the whole range of calcareous grasslands at the regional scale. The aim of this study was (i) to provide a synthesis of the floristic variation of calcareous grasslands of the Calestienne natural region using a uniform methodology; (ii) to relate floristic variation to environmental variables, and (iii) to characterize the specific diversity of the different grassland communities that occur in the study region. Seven different communities were identified with the TWINSPAN method. The originality of the grasslands on calcareous and calcareo-siliceous rocks was statistically confirmed. Significant differences for environmental variables were identified among the seven communities by a MANOVA. Main differences between communities were related to xericity and pH, although a north-south gradient was also identified. More xeric grasslands were located in the southern part of the study region while northern part was occupied by more mesophilous grasslands. Multiple regressions were used to describe the influence of the environmental conditions on plant species richness. The most mesophilous grasslands appeared to be the most species-rich while soil acidity negatively affected species richness.