The gentians (Gentianaceae, Asteridae) form a morphologically and ecologically diverse group of plants with interesting phytochemical and medicinal properties. The family contains six tribes, but phylogenetic relationships among the three more derived tribes (Gentianeae, Helieae, and
Potalieae) are considered ambiguous. In an attempt to resolve alignment ambiguity and provide better scientific support for relationships, the internal transcribed spacer (ITS) of the nuclear ribosomal DNA was aligned for several gentian sequences with reference to the ITS secondary structure
proposed for Anvillea radiata (Asteraceae) using Dynalign. We present here the first secondary structure of ITS for Gentianaceae, which may guide alignment of ITS data in other asterids taxa. The study included 42 species, with representatives from tribes Chironieae, Gentianeae, Helieae,
and Potalieae, and with the majority of species from Potalieae. Phylogenetic analyses based on secondary structure alignment using parsimony and Bayesian methods provided additional support for Gentianeae plus Helieae as the sister group to Potalieae. Prepusa (Helieae) showed ambivalent
associations. Within tribe Potalieae, the subtribes Potaliinae and Lisianthiinae were supported as monophyletic. Bisgoeppertia (Chironieae) was placed unambiguously inside Lisianthius (Potalieae-Lisianthiinae) in all analyses. Neurotheca (Potalieae-Faroinae) was positioned
as sister to Lisianthiinae, making subtribe Faroinae paraphyletic.