Creeping bentgrass (Agrostis stolonifera L.) is the premier turfgrass species used on golf course putting greens, fairways, and tees because of its hardiness, growth and aesthetic characteristics and ability to tolerate low mowing. The genus Agrostis is a polyploid series with genome numbers ranging from 2X to 10X. Creeping bentgrass is a highly outcrossing allotetraploid (2n=4x=28) with the genome designation A2A2A3A3. The diploid species possessing the A2 and A3 subgenomes of creeping bentgrass are currently unknown. One of the difficulties in determining the diploid progenitors of the cultivated bentgrasses is the lack of accurate ploidy determinations for many of the Agrostis accessions maintained in the National Plant Germplasm System (NPGS). The largest U.S. collection of publicly available Agrostis germplasm is housed at NPGS; as of April 19, 2009 there were 368 Agrostis accessions available representing 25 distinct species. The objective of this research was to assess the ploidy level and the amount of genetic diversity present in a subset of the NPGS Agrostis germplasm. In the present study, 75 Agrostis accessions representing 15 distinct species along with two Apera and four Polypogon accessions were screened using flow cytometry and 1,309 Miniature Inverted repeat Transposable Element (MITE) display DNA markers. Cluster analysis clearly separated the common turf-type Agrostis species into distinct groups. The inclusion of previously understudied species within these groups offers insights into the genomic origins of creeping bentgrass that may be useful in future breeding efforts. In addition, these data suggest a narrowing of the genetic diversity within cultivated creeping bentgrasses.