Festuca hallii [(Vasey) Piper] (plains rough fescue) is a dominant native grass species in the Fescue Prairie region of North America that has undergone dramatic range reduction in the past century. This research is undertaken to address the related issues associated with the effectiveness of sampling in capturing genetic diversity, the influence of habitat fragmentation on genetic variation, the geographic variation of seed germination characteristics, and the comparative genetic variation of differential germination. It was found that the tiller samples revealed slightly larger among-population variation than the seed samples. The fescue plant was genetically diverse, as revealed by the proportion of polymorphic bands, the mean band frequency, and the within-population variation. The genetic variation was not highly differentiated with only 6.5% of the total AFLP variation residing among populations. Mantel test revealed a significant correlation between genetic and geographic distances and a spatial autocorrelation up to 60 km among populations was detected. Base temperatures (Tb, minimal or base temperature permitting germination) of the 15 populations fell into a narrow range within 2.2°C with an average of 1.1°C. High final germination percentage was reached at a wide temperature range from 5 to 20°C with the highest germination percentage at 10°C. Germination rate index increased with increasing temperature from 5 to 20°C. Tb was positively correlated with latitude and negatively with longitude and the thermal time requirement for 50% germination was negatively correlated with latitude. The AFLP variation and germination responses were significantly associated with environmental attributes related to moisture, indicating local adaptation. However, the AFLP variation and germination was not significantly associated with the estimated population size and geographic distance to the nearest neighbor, suggesting that fragmentation has not generated considerable genetic and germination impact on the fescue populations. Marked differences in estimates of mean band frequency were observed for various groups of germinating seeds under different test temperatures. Comparisons of AFLP variation among 27 groups of seeds representing population, germination timing and test temperature indicates seed genotypes respond slightly differently to environmental variation, resulting in significant but small impact of germination timing and temperature on the genetic diversity of populations. These findings are significant not only for understanding and predicting the ecological adaptation of the species, but also for formulating effective restoration strategies for remnant populations