Urban areas have developed under anthropogenic impact and hence feature strongly altered environmental conditions that influence today’s plant species distribution. Generally, urban areas are rich in plant species, but on smaller scales richness can vary considerably. Our study aim is to identify areas of high species richness and to assess richness distribution patterns in the city of Hamburg by analyzing a floristic mapping dataset on the scale of 1 km2. Differences in plant species richness were analyzed between three urbanization zones. With multiple regression analyses, we tested effects of urban structure, habitat, and environmental conditions on the distribution of species richness measures. Total species richness per 1 km2 was 274 ± 60 on average and differed only slightly between the urbanization zones. It increased with habitat diversity and decreased with Ellenberg indicator values (EIV) for nutrients (multiple R2 = 0.31). Proportion of non-native species increased with mean annual temperature and decreased with EIVs for moisture (multiple R2 = 0.72), while proportion of endangered species increased with EIVs for moisture and decreased with EIVs for nutrients (multiple R2 = 0.66). Proportion of thermophilic species (multiple R2 = 0.58) could be explained by mean annual temperature. The emerging patterns probably differ from those in other cities due to the central port harboring a particular flora. Besides the expected high proportions of non-native species, high proportions of endangered species were also found in this area. Our results contribute to identifying drivers of biodiversity in cities and can thus be used to develop measures for the conservation of urban biodiversity.