Timing of seed germination influences plant lifetime fitness and can affect the ability of plant populations to colonize and persist in changing environments. However, the genetic variation of the seed germination response remains poorly understood. The amplified restriction fragment polymorphism (AFLP) technique was applied to characterize the genetic variation of germinated seeds collected from three Festuca hallii populations in the Canadian prairie. Three subpopulations with early, intermediate and late germination were identified from each population, based on germination tests at 10, 15 and 20°C in controlled growth chambers. Three AFLP primer pairs were employed to screen a total of 540 assayed seedling samples and 188 polymorphic AFLP bands were scored for each sample. None of the assayed AFLP bands were significantly associated with seed germination, but marked differences in estimates of mean band frequency were observed for various groups of germinating seeds under different test temperatures. Partitioning of the total AFLP variation showed that 5.9% AFLP variation was present among seeds of the three populations, 0.3% among seeds of three germination subpopulations, and 0.5% among seeds grouped for germination temperature. Genetic differentiation was significant among 27 groups of seeds representing population, germination timing and test temperature. Subpopulations with early and intermediate germination shared similar genetic backgrounds and were genetically differentiated from the late germination subpopulation. These results indicate that seed genotypes respond slightly differently to environmental variation, resulting in significant but weak genetic differentiation in the germination of F. hallii seeds. Implications for plant establishment and fescue restoration are discussed.