Recently, it has been suggested that internal transcribed spacer (ITS) sequences are under selective constraints to preserve their secondary structure. Here, we investigate the patterns of the ITS nucleotide and secondary structure conservation across the Passiflora L. genus to evaluate the potential use of secondary structure data as a helpful tool for the alignment in taxonomically complex genera. Considering the frequent use of ITS, this study also presents a perspective on future analyses in other plant groups. The ITS1 and ITS2 sequences presented significant differences for mean values of the lowest energy state (LES) and for number of hairpins in different Passiflora subgenera. Statistical analyses for the subgenera separately support significant differences between the LES values and the total number of secondary structures for ITS. In order to evaluate whether the LES values of ITS secondary structures were related to selective constraints, we compared these results among 120 ITS sequences from Passiflora species and 120 randomly generated sequences. These analyses indicated that Passiflora ITS sequences present characteristics of a region under selective constraint to maintain the secondary structure showing to be a promising tool to improve the alignments and identify sites with non-neutral substitutions or those correlated evolutionary steps.