The Mason Valley cholla, Cylindropuntia ×fosbergii (C. B. Wolf) Rebman, M.A. Baker & Pinkava, is the putative hybrid of C. bigelovii (Engelm.) F. M. Knuth and some other species of Cylindropuntia. We used AFLPs to screen chollas of the Anza-Borrego Desert in southern California to test this hypothesis of hybrid origin and identify the parental species involved. Other species scrutinized as potential parents include C. echinocarpa (Engelm. & J. M. Bigelow) F. M. Knuth, C. ganderi (C. B. Wolf) Rebman & Pinkava, C. californica var. parkeri (J. M. Coulter) Pinkava, and C. wolfii (L. D. Benson) M.A. Baker. Patterns of band sharing clearly testify to the close relationship between C. ×fosbergii and C. bigelovii. None of the other species screened came close to that level of similarity. Moreover, the numbers of total loci and unique loci in C. ×fosbergii do not meet the expectations of a hybrid taxon. We propose the alternative hypothesis that C. ×fosbergii is the sister species of C. bigelovii.