Abstract Background Paspalum plicatulum is a perennial rhizomatous grass with natural diploid and polyploid cytotypes. It is a member of Plicatula, which has historically been recognized as a highly complex group containing species of ecological, ornamental and forage importance. The complex nature of the P. plicatulum genome makes it a challenging species for genetic research. This study aimed to develop and characterize microsatellite molecular markers in P. plicatulum and to evaluate their transferability to other Plicatula group species. Findings Microsatellite sequences were identified from three enriched libraries from P. plicatulum . Specific primers were designed, and 25 displayed polymorphism when screened across 48 polyploid Paspalum spp. genotypes. The number of bands per locus ranged from 2 to 17, with a mean of 8.65. Private bands for each species were identified; the highest number of private bands was observed for P. plicatulum in 52% of the loci analyzed. The mean polymorphism information content of all loci was 0.69, and the mean discriminatory power was 0.82. Microsatellite markers were satisfactorily cross-amplified for the eight tested Plicatula-group Paspalum species, with P. atratum exhibiting the highest transferability rate (89.86%). STRUCTURE and Discriminant Analysis of Principal Components separated accessions into three groups but did not reveal separation of the accessions according to species. Conclusions This study describes the first microsatellite markers in P. plicatulum , which are polymorphic, efficient for the detection and quantification of genetic variation, and show high transferability into other species of the Plicatula group. This set of markers can be used in future genetic and molecular studies necessary for the proper development of conservation and breeding programs. Private bands within the markers can be used to assist in species identification.