BackgroundThe ability to compare RNA secondary structures is important in understanding their biological function and for grouping similar organisms into families by looking at evolutionarily conserved sequences such as 16S rRNA. Most comparison methods and benchmarks in the literature focus on pseudoknot-free structures due to the difficulty of mapping pseudoknots in classical tree representations. Some approaches exist that permit to cluster pseudoknotted RNAs but there is not a general framework for evaluating their performance.ResultsWe introduce an evaluation framework based on a similarity/dissimilarity measure obtained by a comparison method and agglomerative clustering. Their combination automatically partition a set of molecules into groups. To illustrate the framework we define and make available a benchmark of pseudoknotted (16S and 23S) and pseudoknot-free (5S) rRNA secondary structures belonging to Archaea, Bacteria and Eukaryota. We also consider five different comparison methods from the literature that are able to manage pseudoknots. For each method we clusterize the molecules in the benchmark to obtain the taxa at the rank phylum according to the European Nucleotide Archive curated taxonomy. We compute appropriate metrics for each method and we compare their suitability to reconstruct the taxa.