The cotton mouse (Peromyscus gossypinus) is on the northern periphery of its range in southwestern Kentucky, southeastern Missouri, and southern Illinois. Little information is available on the life history of cotton mice in Illinois, in part because of difficulty in differentiating them from white-footed mice (P. leucopus). Current identification is often based on lethal sampling, including collection of internal tissues for allozyme electrophoresis or measurement of skull characters. Here we describe a reliable, non-lethal method for distinguishing between cotton mice and white-footed mice using a diagnostic allozyme locus, glucose-6-phosphate isomerase (GPI-1) from toe-clips. This technique will enhance conservation efforts by making identification of P. gossypinus and P. leucopus easier in areas of sympatry.