Pseudopimelodus is a Neotropical genus of bumblebee catfish, composed of four valid species occurring in both trans - and cis -Andean rivers of South America. The orogeny of the Andes has led to diversification in the genus Pseudopimelodus in Colombia. This study analyzed partial sequences of mitochondrial cox1 and nuclear rag2 genes to test the hypothesis that the species, nominally recognized as P. schultzi and P. bufonius in Colombia, correspond to more than two different evolutionary lineages. Results indicate high levels of genetic divergence among individuals of nominal P. schultzi and P. bufonius, from trans - and cis -Andean basins in Colombia. In addition, five divergent lineages of Pseudopimelodus were confidently delimited by using a single-locus species-discovery approach and confirmed by species tree analyses. Additionally, molecular-clock dating showed that most diversification processes in Pseudopimelodus took place during the Miocene, when Andean tectonic evolution was occurring in northwestern South America. The present study provides, for the first time, phylogeographic insight into this Neotropical genus.