Abstract: Switchgrass is an important bioenergy plant. In this study, the genetic diversity of 65 samples of tetraploid Alamo and mutated octoploid Alamo (using colchicine) was assessed using expressed sequence tag-simple sequence repeat (EST-SSR) and intron-targeted intron-exon splice junction (IT-ISJ) markers. The results showed that EST-SSR primers amplified 245 fragments; the percentage of polymorphic loci was 99.59%, the number of alleles (Na) was 1.9878, the effective number of alleles (Ne) was 1.1718, Nei’s gene diversity (H) was 0.1308, and Shannon’s information index (I) was 0.2388. These genetic parameters for the tetraploid Alamo were as follows: 226 fragments; 88.93% polymorphic loci; Na, 1.8816; Ne, 1.1823; H, 0.1349; and I, 0.2398. These values were higher than those obtained for octoploid Alamo: 217 fragments; 92.24% polymorphic loci; Na, 1.9184; Ne, 1.1652; H, 0.1228; and I, 0.2234. The IT-ISJ primers amplified 165 fragments and the percentage of polymorphic loci was 96.36%, Na was 1.9878, Ne was 1.5777, H was 0.3354, and I was 0.4999. These parameters for tetraploid Alamo were as follows: 145 fragments; 94.55% polymorphic loci; Na, 1.9333; Ne, 1.6366; H, 0.3602; and I, 0.5280. These values were higher than those obtained for octoploid Alamo: 139 fragments; 84.24% polymorphic loci; Na, 1.7515; Ne, 1.4337; H, 0.2573; and I, 0.3864. A clustering analysis with the unweighted pair-group method with arithmetic means did not clearly separate 68 samples of tetraploid Alamo and octoploid Alamo, which had a low level of genetic differentiation. An analysis of molecular variance showed that 94.78% and 80.76% of genetic diversity occurred within ploidy levels, while 5.22% and 19.24% of genetic diversity occurred between ploidy levels.