Including RNA secondary structures improves accuracy and robustness in reconstruction of phylogenetic trees. It is possible to simultaneously infer alignments and phylogenies on the primary sequence and the secondary structure information. For the internal transcribed spacer 2 (ITS2), a phylogenetic RNA transcript marker, two different structure conformations (I or Y shape for helix I) were published for Scenedesmaceae, and a third appeared in the ITS2 database. We contrast the effects on phylogenetic tree reconstruction of different structure sets for a small scenedesmacean subset, using neighbour-joining, maximum parsimony and, for the first time, maximum likelihood, on sequence-structure alignments. Generally our study supports inclusion of secondary structure information. However, we found that any of the three structure conformations is equally fit for phylogenetic studies, but prefer the I shape for helix I. Moreover, our results enable us to give general recommendations on how to build a phylogenetic tree using ITS2 sequence–structure alignments, including different methods to obtain the secondary structures. Thus, we hope to provide a valuable contribution not only for scenedesmacean ITS2 phylogeny, but also for other approaches using RNA transcript markers.