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. Identifying annual ryegrass contamination in perennial ryegrass seed lots has been of major interest in the seed industry for many years. We are developing a multiplexed test based on sequences of flowering- and vernalization-related genes to predict growth habit from samples of ryegrass seed or seedlings. The long-term goal of the project is to develop a testing protocol which can be implemented in commercial grass seed testing laboratories. Subsets of 400 seeds from each seed lot are tested for seedling root fluorescence (SRF), a trait indirectly related to the annual growth habit. DNA is extracted from each SRF seedling, along with nonfluorescent controls, and the samples are screened with a panel of molecular markers, including those developed from single nucleotide polymorphisms (SNP) identified in the genes LpCO and LpID1. LpCO, a putative transcription factor, is similar to the CONSTANS-like homolog ZCCT-1 identified in winter wheat that plays a central role in the vernalization response. LpID1 is a homolog of the gene Indeterminate, shown 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 an SNP marker in LpVrn-1 on linkage group 4, syntenic to the region of the Triticeae chromosome 4 containing the primary vernalization response gene, Vrn-1. PCR-based tests for the isozymes pgi-2 and sod-1, shown to predict annual-type growth habit better than a grow-out test are also being developed. 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.