Urbanization significantly alters natural ecosystems, and its rate is only expected to increase globally as more humans move into urban centers. Urbanized landscapes are often highly fragmented. Isolated populations within these fragments may adapt in response to novel urban ecosystems, but few studies have found strong evidence of evolutionary responses in urban environments. We used multiple genome scan and genotype-environment association (GEA) approaches to examine signatures of selection in transcriptomes from urban white-footed mice (Peromyscus leucopus) in New York City. We scanned transcriptomes from 48 P. leucopus individuals from six environmentally heterogeneous locations (three urban and three rural) for evidence of rapid local adaption in isolated urban habitats. We analyzed 154,770 SNPs and identified patterns of genetic differentiation between urban and rural sites and signatures of selection in a large subset of genes. Neutral demographic processes can create allele frequency patterns that are indistinguishable from positive selection. We accounted for this by simulating a neutral SNP dataset under the inferred demographic history for the sampled P. leucopus populations to serve as a null model when choosing outliers. We annotated the resulting outlier genes and further validated them by associating allele frequency differences with environmental measures of urbanization, percent impervious surface and human population density. The majority of candidate genes were involved in metabolic functions, especially dietary specialization. A subset of these genes have well-established roles in metabolizing lipids and carbohydrates, including transport of cholesterol and desaturation of fatty acids. Our results reveal clear genetic differentiation between rural and urban sites that likely resulted from rapid local adaptation in urbanizing habitats. The specific candidate loci that we identified suggest that populations of P. leucopus are using novel food resources in urban habitats or locally adapting through changes in their metabolism. Our data support the idea that cities represent novel ecosystems with a unique set of selective pressures.