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Buffalograss [Buchloe dactyloides (Nutt.) Engelm.] has the potential for increased use as a turfgrass species due to its low maintenance and water conservation characteristics. This study was conducted to estimate diversity and relationships among naturally occurring buffalograss genotypes based on the nuclear genome, using sequence-related amplified polymorphism (SRAP) markers. The 56 genotypes studied represented five ploidy levels collected from diverse geographic locations in the North American Great Plains. In addition, blue grama [Bouteloua gracilis (H.B.K.) Lag. Ex Steud.] and perennial ryegrass (Lolium perenne L.) were included as outgroups. Twenty-five combinations of forward and reverse primers were used. Ninety-five intensively amplified markers were scored and used to infer diversity and relationships among the genotypes. All buffalograss genotypes were discriminated from each other with similarity values ranging from 0.70 to 0.95. Principal component analysis (PCA) suggested that the 56 genotypes could be reduced to 50 due to high similarity levels among some of the genotypes. The distance between buffalograsses, blue grama, and perennial ryegrass were consistent with current taxonomical distances. This research indicates that SRAP markers can be used to estimate genetic diversity and relationships among naturally occurring buffalograss genotypes.