Ambisense viruses are negative-sense single-stranded RNA viruses that use a unique expression strategy. Their genome contains at least one ambisense RNA segment that carries two oppositely oriented reading frames separated by an intergenic region. It is believed that a structural RNA element within the intergenic region is involved in transcription termination. However, a general overview over the structural repertoire of ambisense intergenic regions is currently lacking. In this study we investigated the structural potential of the intergenic regions of all known ambisense viruses and compared their structural repertoire by structure-guided clustering. Intergenic regions of most ambisense viruses possess a high potential to build stable secondary structures and many viruses share common structural motifs in the intergenic regions of their ambisense segments. We demonstrate that (i) within the phylogenetic virus groups sets of conserved functional structures are present, but that (ii) between the groups conservation is low to non-existent. These results reflect a high degree of freedom to regulate ambisense transcription termination and also imply that the genetic strategy of having an ambisense RNA genome has evolved several times independently.