Perennial ryegrass (Lolium perenne) is a widely used forage species that is often sold as a blend of several cultivars. For example BG34 is a mixture of the cultivars Barlet, Barmaco, Barnhem, and Mara in the proportions of 40, 25, 25 and 10%, respectively. Limited information is available on the stability of these blends after sowing. The objectives of this study were to assess the suitability of SSR markers for ryegrass cultivar differentiation, and to examine the genetic structure of BG34 ryegrass populations. We characterized 50 individuals of each of 2 lines of the four cultivars of BG34, using three SSR markers. Individuals were correctly allocated to lines and cultivars with 80.9-86.7% accuracy using discriminant analysis. There were distinct differences between cultivars, and in some cases, lines-within-cultivars. We also collected 100 ryegrass plants from 5-year old fields of BG34 from three Ohio dairy farms, and compared their SSR profiles with the reference populations. One field remained similar to the cultivar mixture in the original sowing. Change of genetic structure was found for two fields, with increases in the proportion of Mara and Barmaco, and decreases Barlet or Barnhem depending on field. Overall, SSR was proven to be highly effective for differentiating among cultivars and lines.