Native to Central America, papaya (Carica papaya) is one of the most cultivated fruit crops in the tropical areas of the world. Genetic diversity analyses are an important aspect of conservation of plant genetic resources. In the island of Puerto Rico, where papaya has been consumed for centuries, knowledge on the genetic diversity of papaya is lacking. Therefore, 162 papaya accessions were evaluated using 23 simple sequence repeat (SSR) markers. Of these accessions, 139 were farmer-held samples from Puerto Rico, 13 were U.S. Department of Agriculture (USDA) repository samples, and 10 were commercial varieties. A total of 214 alleles were identified with a mean observed heterozygosity (Ho) of 0.219. Inbreeding coefficient (F) was 0.565, and when evaluating the population structure of these accessions, 2 groups (k = 2) were identified. Unweighted pair group method with arithmetic mean (UPGMA) dendrogram showed no geographical organization within the unknown Puerto Rican samples. This assessment provides an extensive record of the genetic diversity of papaya in Puerto Rico which can contribute to breeding strategies and to the conservation of papaya genetic resources in the Caribbean.