We review the trans-Andean species of Ancistrus from Panama, Colombia and Venezuela. Based on analyses of meristic, morphometric and pigmentation pattern data of preserved specimens, eight of sixteen species reported from this region are considered valid and two new species are described. Here we review Ancistrus chagresi Eigenmann & Eigenmann 1889 from both slopes of central Panama; A. centrolepis Regan 1913 from Pacific slopes of eastern Panama and western Colombia; Ancistrus caucanus Fowler 1943, from the Magdalena River drainage in northern Colombia; Ancistrus martini Schultz 1944, from the Lake Maracaibo Basin of Venezuela and Colombia. Ancistrus galani Pérez & Viloria 1994, from a cave in the Lake Maracaibo Basin of Venezuela is considered valid but was not examined. Ancistrus tolima new species is described from the upper Magdalena River drainage and Ancistrus vericaucanus new species is described from the upper Cauca River drainage. Ancistrus gymnorhynchus Kner 1854 and A. falconensis Taphorn, Armbruster & Rodriguez-O. 2010 were treated previously. One specimen of A. clementinae Rendahl 1937 from the Pacific coast of Ecuador was examined, it is considered a valid species. A key for identification and geographical ranges are provided.