The aim of this study was to investigate the floristic composition and diversity of seven urban habitat types in 24 Serbian cities with different climatic affiliation. In each of the 24 cities, we selected 1 ha plots representing a habitat from one of the following groups: square, boulevard, residential area with compact and with open building pattern, city park, and sites with early and mid-succession vegetation stages. All vascular plant species that occur spontaneously in these plots were observed. Data on the main climatic characteristics were collected for each plot, and data on the life forms were obtained for each species recorded. Diagnostic species were identified for each habitat type analyzed, and alpha, beta and gamma diversity were calculated. A total of 674 taxa were recorded in the studied area. Significant differences were observed in habitats by diagnostic species and by life form representation. The lowest alpha and gamma diversity and the dominance of therophytes were observed in habitat types with intensive anthropogenic impact, whereas the highest number was recorded in mid-successional sites and residential areas with a compact building pattern. The analysis showed that habitat type influences species composition much more than climate.