Anopheles cruzii is the main vector of human and simian malaria in the Brazilian Atlantic Forest. This biome, which is an important hotspot of malaria transmission, has suffered fragmentation and deforestation as a result of urban expansion. Fragmentation and deforestation occur continually in the south of the city of São Paulo, Brazil, and findings of An. cruzii in the peridomicile have consequently become more frequent in this part of the city. Although An. cruzii is of considerable epidemiological importance, the impact of urbanization on the microevolution of this species in this malaria-endemic region has not been investigated to date. In this study, we investigated temporal variation in wing shape and size in An. cruzii populations collected in sylvatic, peri-urban and urban areas over a three-year period. Our results show a slight but significant phenotypic variation in all three populations over the study period. Time was a more powerful driver for wing variation than geographic distance. Temporal wing-shape variation appears to be positively associated with urbanization, suggesting that anthropogenic changes in the environment may be a strong driver for wing-shape variation in An. cruzii. Further studies using genetic markers are needed to assess genetic differentiation in these populations.