Urban ecosystems are an increasingly dominant feature of terrestrial landscapes. While evidence that species can adapt to urban environments is accumulating, the mechanisms through which urbanization imposes natural selection on populations are poorly understood. The identification of adaptive phenotypic changes (i.e. clines) along urbanization gradients would facilitate our understanding of the selective factors driving adaptation in cities. Here, we test for phenotypic clines in urban ecosystems by sampling the frequency of a Mendelian-inherited trait—cyanogenesis—in white clover (Trifolium repens L.) populations along urbanization gradients in four cities. Cyanogenesis protects plants from herbivores, but reduces tolerance to freezing temperatures. We found that the frequency of cyanogenic plants within populations decreased towards the urban centre in three of four cities. A field experiment indicated that spatial variation in herbivory is unlikely to explain these clines. Rather, colder minimum winter ground temperatures in urban areas compared with non-urban areas, caused by reduced snow cover in cities, may select against cyanogenesis. In the city with no cline, high snow cover might protect plants from freezing damage in the city centre. Our study suggests that populations are adapting to urbanization gradients, but regional climatic patterns may ultimately determine whether adaptation occurs.