Desmanthus is a genus of forage legumes with high nutritional value, productive potential, and ability to obtain nitrogen in association with diazotrophic bacteria. The use of accurate techniques for genotype identification and characterization is essential for breeding programs. Morphological markers are widely used to know the genetic diversity and the molecular markers are fundamental in these studies. We investigated the genetic diversity among Desmanthus sp genotypes in Pernambuco (Brazilian Northeast State), using morphological traits and ISSR markers. Morphological and molecular characterizations were performed in 18 and 26 accessions, respectively, in plants belonging to the germplasm bank of forage legumes of Universidade Federal Rural de Pernambuco (UFRPE), Academic Unit of Serra Talhada, PE, Brazil. Eight ISSR primers were selected, and 95 loci were generated, with polymorphism of 95.79%. The allele number observed was 1.958, where the effective number was 1.359, and the Nei diversity genetic index was 0.226. About the morphological markers, seed number was the variable that most contributed to the genetic divergence. A large amount of genetic diversity was observed among Desmanthus species, occurring spontaneously in Pernambuco, Brazil. Thus, the variability found in morphological and ISSR markers is importance for the improvement of Desmanthus spp. Our findings showed that 17L, 27L, 25F, 22F, 19S, 13Au, and 28G accessions could be used in breeding programs to explore the maximum genetic divergence.