Perennial ryegrass (Lolium perenne L.) and Italian (or annual) ryegrass (L. multiflorum Lam.) are two of the most widely cultivated grasses used for turf and forage throughout the world. We are developing a multiplex PCR test to detect annual ryegrass contamination in perennial ryegrass seed lots. The long-term goal of the project is to develop a testing protocol that can be implemented in commercial grass seed testing laboratories for samples of ryegrass seed or seedlings. DNA is extracted from leaf tissue of each seedling that shows seedling root fluoresence, along with nonfluorescent controls. The samples are screened with a panel of molecular markers, including those developed based on Lolium flowering-related genes, LpCO and LpID₁. LpCO belongs to a family of putative transcription factors, and is similar to the CONSTANS-like homologs ZCCT-1 and HvZCCT, identified in winter wheat and barley that play a central role in the vernalization response. LpID₁is a homolog of the gene Indeterminateshown to control the transition to flowering in maize. LpID₁ and LpCO have been mapped to ryegrass linkage groups 5 and 7, respectively, in regions associated with vernalization-response QTLs. We have also identified a SNP marker in LpVrn-1 on LG4, syntenic to the region of the Triticeae chromosome 4 containing the primary vernalization response gene, Vrn-1. A panel of 20 ryegrass cultivars (approximately 900 plants) along with ‘Gulf' annual ryegrass controls is being used to refine the test parameters and validate the markers. The occurrence of the LpID₁ marker is strongly correlated with observed morphological groups in the panel, while the LpCO marker appears to be somewhat less predictive. When procedures are optimized, the multiplexed molecular markers will be useful as predictive test in seed testing labs for certifying genetic purity and identity of ryegrass cultivars.