Abstract: Paspalum notatum is a polymorphic species with two accepted varieties, according to the ploidy level. The ploidy level is closely related to the reproductive mode of this species. Usually, diploid cytotypes have sexual reproduction, and tetraploid cytotypes have an apomictic reproduction. Apomixis limits genetic recombination, hindering the development of new cultivars. The identification of sexual plants represents many possibilities in breeding programs. This study aimed to analyze the chromosome number in P. notatum accessions under agronomic evaluation. Ploidy level was determined in 25 accessions. Two ploidy levels were detected, resulting in 21 tetraploid and four diploid accessions. The four diploid accessions originate from Argentina and Uruguay, in a region close to the origin of Pensacola, which is a diploid sexual cultivar of P. notatum. These new diploid accessions may be useful in the development of breeding programs of Paspalum species.