American cutaneous leishmaniasis (ACL) has been characterized as a zoonotic disease. However, peridomestic and domestic transmission have been recorded in at least nine countries in Central and South America. The present study was undertaken to identify the etiologic agent of a peridomestic epidemic of ACL in the Department of Tolima, Colombia. Leishmania isolates were obtained during the diagnosis of 56 patients with ACL who consulted the local leishmaniasis control program in three municipalities in Tolima. Species were identified using monoclonal antibodies and isoenzyme electrophoresis. A total of 53 (94.6%) of 56 isolates were identified as Leishmania (Viannia) guyanensis. Three isolates (5.4%) were identified as L. (V.) panamensis. Leishmania (V.) guyanensis is the probable etiologic agent of the largest epidemic of cutaneous leishmaniasis recorded in Colombia. This species has not previously been reported outside the Amazon and southeastern regions of Colombia, and has not been described in the peridomestic setting or linked with an epidemic.