This article describes a new subspecies of Prepona pylene of western Colombia: Prepona pylene timbiquiensis n. spp., due to a specimen collected by manual capture and baited Van Someren-Rydon traps, in the locality of Saija in the municipality of Timbiqui, department of Cauca. Also we examine and compare to all P. pylene subspecies found in Colombia, based on the review of specimens that are stored in various public and private collections in Colombia. Accordingly we report the synonyms recorded for P. pylene base on the reports found in the literature and the review of the type species described by Hans Fruhstorfer, Henry Walter Bates and William Hewitson deposited in the British Museum in London and the Museum of Zoology, University of Humboldt in Berlin, Germany.