St. Augustinegrass [Stenotaphrum secundatum (Walt.) Kuntze] is a warm-season turfgrass broadly distributed across the southern United States. Here, we investigated genetic diversity and ploidy levels in publicly available plant introductions and cultivars of St. Augustinegrass as an aid to more effective use of these materials in breeding programs. Ploidy assignment of genotypes was problematic in some cases because of a lack of agreement between flow cytometry-inferred ploidy level and chromosome counts indicating that DNA content of higher ploidy genotypes was not a simple multiple of the diploid genome. Cytological investigations indicated five different ploidy levels (diploid, triploid, aneuploid, tetraploid, and hexaploid) with chromosome numbers ranging from 2n = 2x = 18 to 2n = 6x = 54. Principal coordinate and cluster analyses separated genotypes into distinct groups that were mostly congruent with ploidy levels. Moreover, analysis of molecular variance results based on amplified fragment length polymorphism genotyping indicated that 46% of the total variation could be explained by differences between ploidy levels. A clear positive correlation was observed between ploidy level and number of scored bands, with polyploids showing an increased number of bands. Variation in chromosome number is an important source of genetic variation in S. secundatum, and knowledge of the genetic relationships among accessions of this species can be an important consideration for the proper utilization of this germplasm in applied cultivar development. © Crop Science Society of America.