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The Guineagrass (Panicum maximum Jacq.) is one of the most important tropical forage grasses, but genetic knowledge and tools regarding this species are still limited. Therefore, 20 novel polymorphic microsatellite markers were developed, validated, and employed in estimating genetic relationships among 25 P. maximum genotypes selected from a Brazilian germplasm collection. In addition, they were tested for cross-species amplification in four other forage grass species. The number of alleles observed for each locus ranged from 4 to 12 (average 6.7). The values of polymorphism information content (PIC) varied from 0.41 to 0.83 (average 0.61) and the discriminating power (D) ranged from 0.53 to 0.98 (average 0.72). Cross-amplification demonstrated the potential transferability of these microsatellites to four tropical forage grass species. Cluster analysis based on the unweighted pair group method revealed three distinct groups: two clusters consisted of P. maximum genotypes and a third cluster, consisted of the other tropical forage grass species. The data demonstrated that the microsatellites developed herein have potential for germplasm characterization and genetic diversity analysis in P. maximum and other forage grass species.