The taxonomic utility of internal transcribed spacer 2 (ITS2) secondary structures in different plant groups, as well as in Brassicaceae, has been addressed by many studies. Although characterization and applications of ITS2 secondary structures for the members of main Brassicaceae lineages (Lineages I, II, III, and expanded Lineage II) have been studied, the utility of compensatory base change (CBC) species concept has not been the subject of any studies thus far. In the current study, the ITS2 secondary structures of 49 Coluteocarpeae (expanded Lineage II) specimens were investigated to determine relationships among the species. In addition to the utility of the CBC species concept, the availability of hemi-CBC and nonstructural substitutions (NSTs), which are also used for generic and species delimitation, were tested and discussed. A maximum likelihood tree, based on the sequence-structural alignment of the 49 specimens, was constructed to test the different generic assumptions reported by different researchers in the literature. The structural analysis showed that the ITS2 secondary structures of all of the Coluteocarpeae members exhibited a 4-fingered hand model, which was common in the majority of the family members. No CBCs were observed, whereas hemi-CBCs and NSTs were common among the tribe members. Although hemi-CBCs and NSTs were useful for distinguishing most of the Coluteocarpeae species (Noccaea aptera (Velen.) EK. Mey., Noccaea aghrica (P.H. Davis & Kit Tan) M. Firm & Ozudogru, Noccaea fendleri (A.Gray) Holub subsp. glauca (A. Nelson) Al-Shehbaz & M. Koch, Noccaea griffithiana (Boiss.) E.K. Mey., Noccaea rubescens (Schott & Kotschy ex Boiss.) F.K. Mey., etc.), they were not effective for delimitating some problematic species, such as members of Thlaspiceras F.K. Mey. A phylogenetic tree based on the sequence-structural dataset of the ITS2 showed that generic delimitation of Al-Shehbaz was more acceptable due to the fact that Noccaea sensu Al-Shehbaz is monophyletic.