A previous study of spatial genetic structure in brittlebush in the southwestern deserts based on chloroplast DNA variation revealed strongly differentiated populations and statistically significant associations between geography and genetic diversity, presumably associated with Pleistocene climatic oscillations. To expand this work and understand the spatial genetic structure of brittlebush populations more completely, we sought to compare the genetic diversity and structure of chloroplast DNA with that of nuclear ribosomal DNA (internal transcribed spacer) and a low-copy nuclear region developed for the Asteraceae (D22). Here we obtained 192 ITS and 206 D22 sequences from individuals sampled throughout the range and analyzed them with network, population genetic, demographic, hierarchical, spatial, and Bayesian analyses. Although there are differences in the signal present in each genetic region, several large-scale spatial patterns are congruent, including a split between the Sonoran and Mojave Deserts and differentiation of a taxonomic variety from the Cape Region of Baja California. In general, the distribution of genetic variation observed in D22 confirms and even refines patterns previously observed in the chloroplast region. In contrast, there is little to no geographic structure in the genetic variation of ITS, possibly due to the effects of multiple gene copies, reticulation, homoplasy, and concerted evolution. Hierarchical genetic structure differs sharply between nuclear regions and the chloroplast region, and this is likely due to differences not only in the evolution and inheritance of these regions, but also in the dispersal of pollen and seeds among brittlebush populations.