Buchloe dactyloides (Nutt.) Engelm. (buffalograss) is a major component of mixed- and shortgrass prairies of the Great Plains, and is of economic importance both as a forage grass and as a native turfgrass alternative for lawns and golf courses. Like many dominant Great Plains grass species, B. dactyloides comprises an autopolyploid series including diploids, tetraploids, pentaploids, and hexaploids. Preliminary studies indicate that diploids are relatively rare, unlike more broadly distributed higher cytotypes. However, the distribution of these cytotypes is poorly understood. As cytotypes often vary with regards to phenotypically important traits, establishing the distribution of buffalograss cytotypes and evaluating ecological differentiation among them may provide important biological, conservation, and economic insights. This study first aims to establish the geographic distribution of B. dactyloides cytotypes using the locations of both chromosome counted specimens and a set of freshly collected or museum-obtained specimens in which cytotype has been estimated with a genetic approach. Secondly, we assess whether individual cytotypes exhibit differing abiotic tolerances by evaluating both distribution models and statistical analyses of 19 environmental variables. Fifteen microsatellite loci successfully amplified in range of 578 field and herbarium specimens, of which 13 loci were used for cytotype estimation. Microsatellite variation accurately estimated cytotype in 89% of samples from which a chromosome count had been obtained. Diploids and pentaploids were rare, and the common tetraploid and hexaploid cytotypes occupied non-random portions of the buffalograss range. Distribution models and both multivariate and univariate statistical tests establish that the common tetraploid and hexaploid cytotypes are ecologically differentiated.