We report rapid change of morphology and mitochondrial genes in white-footed mice (Peromyscus leucopus) in the Chicago (Illinois, USA) region. We sequenced mitochondrial DNA COX2 from 55 museum skins of white-footed mice caught in the Chicago area since 1855 and from 44 mice recently trapped in the same locations. We found consistent directional genotype replacement at five separate collection locations. We later focused on a single one of these locations (Volo Bog State Natural Area) and sequenced mitochondrial D-loop control region from 58 museum skins of mice collected in 1903–1976 and 32 mice recently trapped there. We found complete and more recent replacement of D-loop haplotypes, apparently occurring between 1976 and 2001. We tested whether these genetic changes were mirrored by changes in morphology by comparing 15 external and cranial traits. We found no significant morphological differences between mice collected in 1903–1976; however, mice collected in 2001–2003 showed 9 of 15 measurements to be significantly changed relative to the earlier samples. Recent mice were longer in total length, with broader, longer noses, and longer but shallower skulls1. Discriminant function analysis allowed for 100% correct classification using these traits. Principal components analysis shows variance over time is well distributed across both external and cranial measures. The sequential replacements of haplotypes and the rapid change of morphology can best be explained by replacement of the regional population with immigrants from genetically distinct neighbouring populations, likely facilitated by the large environmental changes occurring over the time period. Replacement with genotypes from external populations may be a common mechanism of evolution of newly adaptive local forms in an increasingly human-impacted world.