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Twenty eight natural populations of Trifolium riograndense Burkart, an important forage legume from native pastures of the state of Rio Grande do Sul, Brazil, were evaluated for genetic diversity with eight Simple Sequences Repeats (SSR) markers. Chromosome numbers were also determined. The eight markers were polymorphic, with 35 alleles and an average of 4.37 alleles per locus, and Polymorphism Information Content (PIC) between 0.48 and 0.80. Group analysis based on Jaccard´s similarity coefficient separated the 28 accessions in nine groups, with an average genetic similarity of 0.44, indicating a high genetic variability among the populations. No evident relation between genetic distance and geographical origin was detected. The chromosome number of 2n=2x=16 was found in all populations, indicating lack of intraspecific variability for chromosome number in the species. This information on diversity can be used in conservation strategies as well as in genetic breeding programs of this species.