The Urban Heat Island Effect (UHIE) is a globally consistent pressure on species living in cities. Rapid adaptation to the UHIE may be necessary for urban wild flora to persist in cities, but experimental evidence is lacking. Here, we report the first evidence of genetic differentiation in a plant species in response to the UHIE. We collected seeds from common dandelion (Taraxacum officinale) individuals along an urban-rural gradient in the city of Amsterdam (The Netherlands). In common-environment greenhouse experiments, we assessed the effect of elevated temperatures on plant growth and the effect of vernalisation treatments on flowering phenology. We found that urban plants accumulate more biomass at higher temperatures and require shorter vernalisation to induce flowering compared to rural plants. Differentiation was also observed between different intra-urban subhabitats, with park plants displaying a higher vernalisation requirement than street plants. Our results show strong differentiation between urban and rural dandelions in temperature-dependent growth and phenology, consistent with adaptive divergence in response to the UHIE. Rapid adaptation to the UHIE may be a potential explanation for the widespread success of dandelions in urban environments.