Biligetu, B., Schellenberg, M. P. and Fu, Y.B. 2013. Genetic diversity of side-oats grama [Bouteloua curtipendula (Michx.) Torr.] populations as revealed by amplified fragment length polymorphism markers. Can. J. Plant Sci. 93: 1105-1114. Side-oats grama [Bouteloua curtipendula (Michx.) Torr.] is a warm-season grass widely distributed in North America and is considered as an important grass for reclamation and summer forage production in drier regions. This study assessed genetic diversity of nine wild populations of side-oats grama grass, their corresponding balanced multiple-site composite (BMSC), and a population selected for forage type (FT), using the amplified fragment length polymorphism (AFLP) technique. Five AFLP primer pairs were employed to genotype 157 plants, and 312 polymorphic AFLP bands were detected. The frequencies of AFLP bands ranged from 0.01 to 0.99, and averaged 0.39. The AFLP analysis revealed 6% of the total AFLP variation present among the nine wild populations and 94% of variation within populations. The Minto population had the largest within-population diversity, while the FT population the lowest based on AFLP band richness and polymorphic loci. The BMSC population displayed significant genetic differentiation from the wild populations, but still captured substantial genetic diversity. Bayesian cluster analysis using BAPS and STRUCTURE programs revealed three and four optimal clusters of populations that explained 9.8 and 9.5% of the total AFLP variation, respectively. A genetic clustering of individual plants displayed no clear genetic separations among wild, FT, and BMSC populations, but the FT population showed some level of genetic shift, indicating the initial impact of artificial selection. These findings are significant for our understanding of the genetic diversity of side-oats grama. Large genetic variation present within populations provides a potential for further genetic enhancement.