The ITS region (nrDNA) was sequenced for 90 representatives of the Cactaceae subfamily Opuntioideae, and 10 outgroup taxa from the subfamilies Cactoideae, Maihuenioideae and Pereskioideae, and the family Portulacaceae. A consensus of over 1000 shortest trees found by parsimony analysis of these sequences support the following relationships: The Opuntioideae form a well-supported sister clade to the remaining Cactaceae. The Pereskioideae form a basal grade to the subfamilies Mahuenioideae and Cactoideae, which are monophyletic sister groups. Maihueniopsis is sister to the remaining Opuntioideae, which includes two clades marked by terete or flattened stem morphology. Relationships among the species of Austrocylindropuntia, Cylindropuntia, Grusonia, and Quiabentia are not resolved. Although Cumulopuntia and Tephrocactus are well supported as monophyletic groups, the relationships of these genera within the terete-stemmed Opuntioideae are unclear. Pereskiopsis may be sister to a clade comprised of Cylindropuntia and Grusonia, although this topology lacks strong statistical support. Grusonia pulchella is sister to Cylindropuntia s.l. The flat-stemmed Opuntioideae form a well-supported clade. A clade consisting of Tunilla and Miqueliopuntia is sister to the remaining platyopuntioids. The genera Consolea and Nopalea are well supported as monophyletic, but appear to be nested within Opuntia s.s. Terete stems appear to be homoplastic. These data suggest a South American origin for both terete- and flat-stemmed Opuntioideae.

Key words: Cactaceae, Opuntia, Opuntioideae