Characterizing and inferring the buffalograss [Buchloe dactyloides (Nutt.) Engelm.] genome organization and its relationship to geographic distribution are among the purposes of the buffalograss breeding and genetics program. This buffalograss study was initiated to: (1) better understand the buffalograss ploidy complex using various marker systems representing nuclear and organelle genomes; (2) determine whether the geographic distribution was related to nuclear and organelle genome variation; and (3) compare the genetic structure of accessions with different ploidy levels. The 20 buffalograss genotypes (15 individuals from each genotype) that were studied included diploid, tetraploid, pentaploid, and hexaploid using nuclear (intersimple sequence repeat (ISSRs), simple sequence repeat (SSRs), sequence related amplified polymorphism (SRAPs), and random amplified polymorphic DNA (RAPDs)) and cytoplasmic markers (mtDNA and cpDNA). There was a significant correlation between the ploidy levels and number of alleles detected using nuclear DNA (ISSR, SSR, and SRAP, r=0.39, 0.39, and 0.41, P < 0.05, respectively), but no significant correlation was detected when mitochondrial (r=0.17, P < 0.05) and chloroplast (r=0.11, P < 0.05) DNA data sets were used. The geographic distribution of buffalograss was not correlated with nuclear and organelle genome variation for the genotypes studied. Among the total populations sampled, regression analysis indicated that geographic distance could not explain genetic differences between accessions. However, genetic distances of those populations from the southern portion of buffalograss adaptation were significantly correlated with geographic distance (r= 0.48, P < 0.05). This result supports the hypothesis that genetic relationship among buffalograss populations cannot be estimated based only on geographic proximity.