Urban areas exert a significant influence on plant species assemblages. The mosaic of different urban land uses is reflected in the distribution patterns of different plant groups. Here we present the results of the first systematic and detailed floristic survey of the city of Brno, Czech Republic. We studied the flora of Brno from 2011 to 2021, and recorded all spontaneously occurring species in grid cells of 1.3 × 1.5 km. Our dataset includes 1,492 taxa found in the city, classified by their origin, residence time, invasion status, index of ecological specialization, and threat status in the Czech flora. Of these, 902 are native, 205 archaeophytes and 339 neophytes. The remaining 46 species with unknown status are probably remnants of cultivation or newly introduced species. Of the total list of species, 255 species are classified as threatened or near threatened in the Czech Republic. We analysed the effect of seven land-use categories on the proportions of these plant groups and found significant differences in the distribution of individual plant groups within the city. The proportions of plant groups except for threatened species reflected the proportions of individual land-use categories in the grid cells, although the strength and direction of these responses differed among plant groups. Native plant species richness was high in grid cells where forests predominate and the level of urbanization is low. In contrast, the proportion of archaeophytes and neophytes was much lower in the grid cells with a high proportion of forests. While archaeophytes predominated in the lowlands with agricultural land use, neophytes were more common in the central built-up areas of the city. To document the current distribution of all taxa found we supplement this study with a series of maps.