Ryegrass species are among the most important species in sown pastures, turf settings, and weed populations worldwide. Perennial ryegrass (Lolium perenne L.) is an outcrossing, wind-pollinated grass. Recent research has demonstrated the feasibility of developing transgenic perennial ryegrass varieties. In order to model the consequences of gene flow from transgenic grass genotypes in a field situation, the model non-transgenic trait of fertility among autotetraploid genotypes was chosen. Gene flow over distance and direction from a donor plot to surrounding sexually compatible recipient plants was studied. Reproductive isolation was achieved through the fertility barrier that arises between tetraploid and diploid ryegrass genotypes, despite the presence of diploid plants in a meadow situation. Fertility was used as an indication of effective gene flow over distance and direction. Measures of the fertility of recipient plants included total seed production (TSP), floret site utilisation (FSU), and relative fertility of recipient plants as a percentage of those within the donor plot (RF%). A leptokurtic distribution for gene flow was identified, with differences in the rate of decline over distance depending on direction. Simple sequence repeat (SSR) polymorphism was used to identify the paternity of progeny plants. The proportional representation of parents among the progeny was not significantly different from that expected due to the numerical representation of the different donor parent genotypes. The results of this research will have important implications for risk analysis prior to the field release of transgenic ryegrasses, fescues, and other pasture grass species, and for seed production in terms of cultivar purity and optimum isolation distance.