Recently, it was determined that representatives of the riboswitch candidates called ykkC and mini-ykkC directly bind free guanidine. These riboswitches regulate the expression of genes whose protein products are implicated in overcoming the toxic effects of this ligand. Thus, the relevant ykkC motif and mini-ykkC motif RNAs have been classified as guanidine-I and guanidine-II riboswitch RNAs, respectively. Moreover, we had previously noted that a third candidate riboswitch class, called ykkC-III, was associated with a distribution of genes similar to those of the other two motifs. Therefore, it was predicted that ykkC-III motif RNAs would sense and respond to the same ligand. In this report, we present biochemical data supporting the hypothesis that ykkC-III RNAs represent a third class of guanidine-sensing RNAs called guanidine-III riboswitches. Members of the guanidine-III riboswitch class bind their ligand with an affinity similar to that observed for members of the other two classes. Notably, there are some sequence similarities between guanidine-II and guanidine-III riboswitches. However, the characteristics of ligand discrimination by guanidine-III RNAs are different from those of the other guanidine-binding motifs, suggesting that the binding pockets have distinct features among the three riboswitch classes.