Populations of Pilosella (Hieracium subgenus Pilosella) at ruderal localities were investigated in an urban area (Prague City) with respect to their distribution, variation in DNA ploidy level/chromosome number and mode of reproduction. The following species, hybridogenous species or hybrids (with ploidy level/chromosome number and mode of reproduction) were found: P. aurantiaca, P. caespitosa (4x, 5x), P. cymosa subsp. vaillantii (5x), P. officinarum (2n = 36, sexual; 2n = 54, sexual; 2n = 63), P. piloselloides subsp. bauhinii (2n = 45, 54; both apomictic), P. piloselloides subsp. praealta (5x; apomictic), P. brachiata (4x; sterile), P. densiflora, P. flagellaris, P. floribunda, P. erythrochrista, P. glomerata (5x; apomictic), P. leptophyton (5x; apomictic), P. rothiana (4x, apomictic), P. setigera, P. visianii (4x; apomictic), P. ziziana (4x, apomictic) and the previously undescribed hybridogenous type P. piloselloides × P. setigera (5x, apomictic). Pilosella visianii is reported from the Czech Republic for the first time. New habitats resulting from highway construction are suitable for Pilosella species. Many previously rare types, such as P. rothiana, can colonize these habitats and spread, not only locally, but also throughout the whole country.