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An eastern woodrat (Neotoma floridana) collected in January 2001 near Bedias, Grimes County, Texas, had extensive lesions of both ears and swollen feet. Impression smears and histologic sections demonstrated the presence of Leishmania in both ears and the one foot that was screened. Polymerase chain reaction screening using species-specific primers detected parasites in both ears and all four feet and indicated the parasites were L. mexicana. The detection of L. mexicana in N. floridana represents a new host record in a new ecologic region and may help explain a human infection acquired outside the previously-known range of the disease. Given the geographic distribution of N. floridana and the two other species of Neotoma found naturally infected, enzootic foci of Leishmania could be present over much of the southern United States.