PREMISE OF THE STUDY: Although numerous phylogenetic studies have been conducted in Cactaceae, whole plastome datasets have not been employed. We used the chollas to develop a plastome dataset for phylogeny reconstruction to test species relationships, biogeography, clade age and morphological evolution. METHODS: We developed a plastome dataset for most known diploid members of the chollas, 42 taxa, as well as for other members of Cylindropuntieae. Paired-end, raw reads from genome skimming were referenced-mapped onto a de novo plastome assembly of one species of cholla, Cylindropuntia bigelovii, which were used to build our plastome dataset that was analyzed using various methods. KEY RESULTS: Our plastome dataset resolved the phylogeny of the chollas, including most inter- and intraspecific relationships. Tribe Cylindropuntieae arose during the early Miocene in southern South America, ca. 18 mya, and is supported as sister to the South American clade Tephrocacteae. The (Micropuntia (Cylindropuntia + Grusonia)) clade most likely originated in the Chihuahuan Desert region around 16 mya, and then migrated into other North American desert regions. Key morphological characters for recognizing traditional taxonomic series (e.g., spiny fruit) in Cylindropuntia are mostly homoplasious. CONCLUSIONS: This study provides the first comprehensive plastome phylogeny for any clade within Cactaceae. Although widespread throughout western North American deserts, the most recent common ancestor of the chollas s.l. likely arose in the Chihuahuan Desert region during the mid-Miocene, with much of their species diversity arising in the early to mid-Pliocene, showing a strikingly similar pattern to other western North American Desert groups.