ABSTRACTViruses in the orderPicornaviralespossess a positive-strand RNA genome that encodes structural proteins (SPs) and nonstructural proteins (NSPs). According to the recent report of the International Committee on Taxonomy of Viruses (ICTV), there are 8 families inPicornavirales, and monopartite picornaviruses in each family exhibit distinct types of genome organizations with rearranged genes coding for SPs and NSPs, namely, TypeI (5′-SPs-NSPs-3′) and TypeII (5′-NSPs-SPs-3′). In the present study, 2 iflaviruses with the 2 genome types were unexpectedly identified in a damselfly host species, suggesting that these 2 genome types coexisted in the same host species, and the families of orderPicornaviralesmight be more complex than previously thought. The consequent systematic homologous screening with all the publicly available picornaviruses successfully revealed a considerable number of candidates rearranged genome types of picornaviruses in various families ofPicornavirales. Subsequently, phylogenetic trees were reconstructed based on RNA dependent RNA polymerase and coat protein, which evidently confirmed the prevalence of the 10 typeII iflaviruses in theIflaviridaefamily. This suggests that genome types may not be relevant to viral taxonomy in this family. However, candidate picornaviruses with reversed genome types in theSecoviridaeandDicistroviridaefamilies require further investigation. All in all, as the number of newly discovered viruses increases, more viruses with non-canonical genome arrangements will be uncovered, which can expand our current knowledge on the genome complexity and evolution of picornaviruses.IMPORTANCEMonopartite viruses in the orderPicornaviralesexhibit distinct genome arrangement of nonstructural proteins and structural proteins for each of the 8 families. Recent studies indicated that at least 4 ifla-like viruses possessed reversed genome organization in the familyIflaviridae, raising the possibility that this phenomenon may commonly present in different families of picornaviruses. Since we discovered 2 iflaviruses with exchanged structural and nonstructural proteins simultaneously in the damselfly, a systematic screening was subsequently performed for all of the current available picornaviruses (1,543 candidates). The results revealed 10 picornaviruses with reversed genome organization in the familyIflaviridae, implying that this phenomenon might prevalence in the orderPicornavirales. These results will contribute to a better understanding for the future study on the genome complexity and taxonomy of picornaviruses.