As the nuclear portion of a multigene analysis of relationships in Orchidaceae, ITS2 and the first 1200 bp of the 26S locus were sequenced in over 60 genera from the family. Alignment of the 26S segment was straightforward, while that of ITS2 was less so. Regions within ITS2 varied with respect to base substitution and indel rate, presumably reflecting secondary structure relationships in the molecule. Equally weighted parsimony analysis of these data yielded two most parsimonious trees, the consensus of which is well resolved. While analysis of the 26S sequences alone gave a moderately resolved tree, addition of the ITS2 data increased resolution and branch support, particularly among closely related groups. Five subfamily-level assemblages are resolved, corresponding to Vanilloideae, Cypripedioideae, Orchidoideae (including Spiranthoideae), and Epidendroideae. Tribal level groups are apparent, and jackknife analysis indicates that while most of these groups are well supported, the relationships among them often are not. This is particularly true among groups within Epidendroideae, where branch lengths are typically short. This is the same pattern seen in other molecular and morphological data sets and is interpreted to reflect a rapid radiation of epidendroid groups. Basal clades in Epidendroideae include Neottieae, Elleanthus, Triphora and Tropidia, with more derived groups including an Asian clade corresponding to Podochileae, as well as a monophyletic Maxillarieae. The patterns are highly congruent with those obtained from plastid and mitochondrial sequence data.

Key words: 26S, ITS, Orchidaceae, phylogeny