The Grijalva River basin is one of the largest hydrological systems in Central America. Despite its importance, an inventory of its ichthyofauna remains incomplete. We provide a systematic checklist of fishes recorded in the Grijalva River basin based on surveys performed from 2002 to 2015. The systematic list includes 92 species, belonging to 13 orders, 25 families and 50 genera, of which 83 species are native and nine are exotics. This checklist includes three range expansions (Profundulus hildebrandi, Poecilia sphenops and Paraneetroplus hartwegi). Fifteen species are endemic to the basin. The most diverse families are Cichlidae and Poeciliidae with 25 and 23 species, respectively. Secondary freshwater fish diversity is high with 65.1%, the remaining species are primary freshwater (13.3%) and peripheral (21.7%) fishes.