Most male date palms available for pollinating different female cultivars have mainly originated from seed propagation resulting in many different local males that represent a source of genetic diversity. Favorable fruit production is related to quality of pollen and its compatibility with a certain female variety. Therefore, the genetic characterization of the male pollen as a superior one for each female cultivar should be the first step to establish an intensive program to produce superior males through different procedures. In this study, the genetic diversity and population structure of 72 male date palm accessions were investigated using 15 microsatellite loci. A total number of 125 alleles was detected with an average of 8.33 alleles per locus. Bayesian model-based clustering analysis indicated the presence of two differentiated endemic male date palm genetic clusters, continental and insular, with the presence of introduced accessions originating from the Middle East. The diversity analysis in the insular region of Tunisia, which had never been performed before, revealed that this germplasm enclosed valuable endemic resources supporting the hypothesis of the presence of wild material. These findings are crucial for identifying interesting genotypes that can be integrated into international coordinated actions of Phoenix dactylifera L. breeding programs as well as for the protection and conservation of valuable resources.