Premise of the study Although autopolyploidy is common among dominant Great Plains grasses, the distribution of cytotypes within a given species is typically poorly understood. This study aims to establish the geographic distribution of cytotypes within buffalograss (Buchloë dactyloides), and to assess whether individual cytotypes exhibit differing ecological tolerances. Methods A range-wide set of 578 B. dactyloides individuals was obtained through field collecting and sampling from herbarium specimens. The cytotype of each sample was estimated by observing allele numbers at thirteen simple sequence repeat loci, a strategy that was assessed by comparing estimated to known cytotype in 79 chromosome-counted samples. Ecological differentiation between the dominant tetraploid and hexaploid cytotypes was assessed with analyses of macro-climatic variables. Key results Simple sequence repeat variation accurately estimated cytotype in 89% of samples from which a chromosome count had been obtained. Applying this approach to samples of unknown ploidy established that diploids and pentaploids are rare, with the common tetraploid and hexaploid cytotypes generally occurring in sites to the north/west (tetraploid) or south/east (hexaploid) portions of the species range. Both MANOVA and niche modeling approaches identified significant but subtle differences in macro-climatic conditions at the set of locations occupied by these two dominant cytotypes. Conclusions Incorporating chromosome count vouchers and cytotype-estimated herbarium records allowed us to perform the largest study of cytotype niche differentiation to date. Buffalograss cytotypes differ greatly in frequency, the common tetraploid and hexaploid cytotypes are non-randomly distributed, and these two cytotypes are subtly ecologically differentiated.