Morphometric measurements or biochemical methods are often required to differentiate deer mice, Peromyscus maniculatus (Wagner, 1845), from white-footed mice, Peromyscus leucopus (Rafinesque, 1818), particularly when they are found in sympatry. However, these approaches cannot easily be applied to juveniles, or to degraded or ancient museum specimens. In this paper, we propose a rapid and non-invasive molecular approach to discriminate these cryptic species from one another. This technique relies on species-specific primers designed in the COIII mitochondrial region to amplify fragments of different lengths in each species. The method developed proved useful for the identification of ethanol-preserved, frozen, degraded, or dry museum specimens.