Three subspecies (ssp. tridentata, ssp, vaseyana, ssp. wyomingensis) of big sagebrush (Artemisia tridentata) occupy much of the landscape in western North America. Their habitat range spans much of the cold dry basins and mountains from the northern Baja of Mexico to southern British Columbia of Canada. Over much of this range the subspecies of big sagebrush are the dominant species of the ecosystem providing critical forage and vegetative cover for wildlife. While a dominant species, virtually nothing was known of the genetic structure of big sagebrush until this study. We resequenced amplicons of 48 independent genes reconstructed from EST sequences in 357 samples distributed throughout the range of big sagebrush. Clear phylogenetic distinctions were identified between diploid ssp. tridentata and ssp. vaseyana for many amplicons. However, network analyses show many haplotypes are shared between these subspecies, supporting previous work that diploid subspecies frequently hybridize. Tetraploid populations were polyphyletic including ssp. wyomingensis, showing membership within tridentata and vaseyana subspecies clades. These results suggest tetraploid populations are derived locally as autotetraploid or allotetraploids from diploids of tridentata and vaseyana subspecies.