Sicydiine gobies are major contributors to Caribbean stream fish biodiversity, and ecosystem functions and services. In the Caribbean, Sicydiine gobies are represented by a single genus, Sicydium, but species-level Sicydium taxonomy and distributions remain unresolved in this region. A previous study posited that four species of Sicydium are present in Puerto Rico, including the recently described Sicydium gilberti; however, that study relied solely on morphological characters, and samples from a limited number of locations. Identification of Sicydium to species in studies with more comprehensive sampling has not occurred due to the impracticality of field identification. In our study, we employed morphological data and Mitochondrial Cytochrome C Oxidase I (mtCOI) genetic sequence data, collected from over 500 Sicydium spp. individuals at 16 broadly distributed sites, to provide an inventory of the species of Sicydium that occur in Puerto Rico. Additionally, we reveal new information on the spatial patterns of distribution, relative abundance, population structure, and observed molecular diversity among Sicydium taxa in Puerto Rico’s river systems. This information is essential to advance ecological and evolutionary knowledge of Caribbean fish assemblages, guide stream fish conservation, and for effective management of fisheries.