Urbanization significantly alters natural ecosystems and has accelerated globally as humans move into dense urban centers. Urban wildlife populations are often highly fragmented by an inhospitable matrix of human infrastructure. Isolated populations may adapt in response to novel urban pressures, but few studies have found evidence of selection in urban environments. We used multiple approaches to examine signatures of selection in transcriptomes from white-footed mice (Peromyscus leucopus) in New York City. We analyzed transcriptomes from 48 P. leucopus individuals from three urban and three rural populations for evidence of rapid local adaption in isolated urban habitats. We generated a dataset of 154,770 SNPs and analyzed patterns of genetic differentiation between urban and rural sites. We also used genome scans and genotype-by-environment (GEA) analyses to identify signatures of selection in a large subset of genes. Neutral demographic processes may create allele frequency patterns that are indistinguishable from positive selection. Thus, we accounted for demography by simulating a neutral SNP dataset under the inferred demographic history for the sampled P. leucopus populations to serve as a null model for outlier analysis. We then annotated outlier genes and further validated them by associating allele frequency differences with two urbanization variables: percent impervious surface and human population density. Many 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 resulted from rapid local adaptation and drift in urbanizing habitats. The specific outlier loci that we identified suggest that populations of P. leucopus are using novel food resources in urban habitats and selection pressures are acting to change metabolic pathways. Our findings support the idea that cities represent novel ecosystems with a unique set of selective pressures.