Guineagrass (Panicum maximum Jacq.) is a forage grass found in tropical and subtropical regions. It is an apomictic and tetraploid species from Africa. The objective of this study was to evaluate the genetic diversity of guineagrass accessions sampled from its regions of origin, which is in Tanzania and Kenya. In this study, a total of 396 accessions were analyzed, and a collection of reproducible and informative microsatellites was developed. Thirty microsatellites were employed to characterize these accessions. A total of 576 clones were sequenced from microsatellite-enriched libraries. Flanking primers were designed for 116 microsatellite loci and screened using a sample of 25 guineagrass accessions. The thirty selected polymorphic microsatellites employed in this study produced a total of 192 bands when evaluated in the 396 P. maximum accessions, with an average of 6.4 bands per microsatellite. Four genetic clusters were identified in the collection using STRUCTURE analysis, and these results were confirmed using AMOVA. The largest genetic variation was found within clusters (65.38%). This study revealed that the collection of accessions from the P. maximum region of origin was a rich source of genetic variability. The geographical distances and genetic similarities among accessions did not indicate a significant association between genetic and geographical variation, supporting the natural interspecific crossing between P. maximum, P. infestum and P. trichocladum as the origin of the high genetic variability and the existence of an agamic complex formed by these three species.