The Cusiana River sub-basin has been identified as a priority conservation area in the Orinoco region in Colombia due to its high species diversity. This study presents an updated checklist and identification key for fishes of the Cusiana River sub-basin. The checklist was assembled through direct examination of specimens deposited in the main Colombian ichthyological collections. A total of 2020 lots from 167 different localities from the Cusiana River sub-basin were examined and ranged from 153 to 2970 m in elevation. The highest number of records were from the piedmont region (1091, 54.0 %), followed by the Llanos (878, 43.5 %) and Andean (51, 2.5 %). 241 species distributed in 9 orders, 40 families, and 158 genera were found. The fish species richness observed (241), represents 77.7 % of the 314 estimated species (95 % CI=276.1-394.8). The use of databases to develop lists of fish species is not entirely reliable; therefore taxonomic verification of specimens in collections is essential. The results will facilitate comparisons with other sub-basins of the Orinoquia, which are not categorized as areas of importance for conservation in Colombia.