Previously, two riboswitch classes have been identified that sense and respond to the hypermodified nucleobase called prequeuosine1 (preQ1). The enormous expansion of available genomic DNA sequence data creates new opportunities to identify additional representatives of the known riboswitch classes and to discover novel classes. We conducted bioinformatics searches on microbial genomic DNA data sets to discover numerous additional examples belonging to the two previously known riboswitch classes for preQ1 (classes preQ1-I and preQ1-II), including some structural variants that further restrict ligand specificity. Additionally, we discovered a third preQ1-binding riboswitch class (preQ1-III) that is structurally distinct from previously known classes. These findings demonstrate that numerous organisms monitor the concentrations of this modified nucleobase by exploiting one or more riboswitch classes for this widespread compound.