Subspecies composition and genetic variation are important to the growth and survival of Largemouth Bass and information on these genetic characteristics is required for proper management of the species. In Puerto Rico, a genetic evaluation is needed to develop management plans for Puerto Rico reservoirs and maintain Florida Largemouth Bass at the Maricao Hatchery. Subspecies composition was characterized among reservoirs and the contemporary Maricao Hatchery broodstock in Puerto Rico, and metrics were compared between subspecies and hybrids among reservoir populations. Genetic variation and structure was determined and compared to Largemouth Bass populations in their native range. Florida subspecies were prevalent in all populations and non-introgressed subspecies seemed to be better adapted to the island than introgressed forms. Genetic diversity was lower in Puerto Rico populations and substantial genetic structure among populations was lacking. These results provide genetic information to guide future management and propagation of Largemouth Bass in Puerto Rico.