We have assessed the utility of morphological and microsatellite markers for tracing field hybridization between Lolium multiflorum and Lolium perenne in cereal-enclosed gene flow plots. The presence of awns on the inflorescence of F1 hybrids was found to be a reliable, but underscoring, indicator of L. multiflorum paternity in L. perenne derived seed as determined by inheritance of species-specific alleles at the microsatellite locus ‘H01 H06’ in these progeny. A positive correlation was evident in the experimental treatment between the number of pollen donor plants in a given plot and the frequency of hybrid F1 seed harvested from pollen receptor plants in that plot. These experiments have established the utility of naturally occurring heritable markers for the measurement of gene flow rates in field Ryegrass populations, with particular significance for risk assessment modeling of potential gene flow from transgenic grass cultivars.