Native prairie grasses of western Canada have the potential for development as turf and forage grass cultivars for semiarid environments. Nuttall’s salt-meadow, or alkali grass (Puccinellia nuttalliana (Shultes) Hitchc.), is a native grass species in North America well known for its salt tolerance. Little information is available about the genetic diversity of natural populations of this species. Understanding the genetic diversity of this species is a prerequisite for developing populations for forage or turf use in western Canada. The objectives of this study were to assess the variation in agronomic/morphological characters and AFLP markers of collections of Puccinellia and identify promising populations and genotypes for turf and forage utilization. A four replicate randomized complete block field nursery of twenty-four collections from western Canada was established in 2010. Plant height, tiller number, crown diameter, dry matter yield, seed yield, and leaf related characters were measured for each collection in the summers of 2011 and 2012. Considerable phenotypic variation was detected among and within the twenty-four populations. Promising populations and genotypes were identified with respect to their superior turf and forage related characteristics. The amplified fragment length polymorphism (AFLP) technique was used to assess the comparative genetic diversity of the collections. Five AFLP primer pairs were employed to screen 15 genotypes from each population, and 185 polymorphic AFLP bands were scored for each sample. Their frequencies of occurrence ranged from 0.02 to 0.99 with a mean of 0.61. The analysis of molecular variance revealed more than 96% of the total AFLP variation resided within populations. Populations were not highly differentiated with only 4% of the total AFLP variation residing among populations. A Mantel test revealed a significant but low correlation between genetic and geographic distances (r=0.293; P=0.024) and non-significant correlation between genetic and phenotypic distances (r=0.070; P=0.282). Implications for P. nuttalliana conservation, germplasm sampling, and cultivar development are discussed.