Diamesinae is one of the eleven subfamilies of Chironomidae, characterized by its preference for cold waters, typically found in mountainous regions. We aimed to study the biodiversity and distribution of two genera of this subfamily found in high-altitude streams in the Andes (Paraheptagyia and Limaya), by combining morphological and molecular analyses. A database with 190 larval specimens of Diamesinae, collected between July and October of 2011 from 20 streams in Colombia, Ecuador, and Peru, at altitudes above 2000 m.a.s.l. was used. Paraheptagyia was found in the three countries, whereas Limaya was only found in Ecuador and Perú. Morphological analyses were based on 105 larvae mountings, using several specimens for each morphospecies, as a result, the antennal ratio (AR) was a valuable differentiator for Limaya, whereas Paraheptagyia could not be distinguished by using morphological characters. The ABGD, ASAP, and bPTP methods were used to delineate operational taxonomic units (OTUs) using 130 Molecular sequences (113 for Paraheptagyia and 17 for Limaya). For the genus Limaya, two OTUs were identified using all methods, one for Ecuador and one for Peru. Seven OTUs were identified for the genus Paraheptagyia. Five of the seven OTUs were found in Peru and the other two were shared between Ecuador and Colombia. The presence of the Huancabamba depression on the border between Ecuador and Peru likely acts as a genetic barrier, limiting gene flow. The difference in branch length between Peruvian and Ecuadorian/Colombian species supports Brundin’s (1966) hypothesis, indicating a south-to-north colonization pattern.