Picornaviruses cause diseases in a wide range of vertebrates, invertebrates and plants. Here, a novel picornavirus was identified by RNA-seq technology from rice plants showing dwarfing and curling symptoms, and the name rice curl dwarf-associated virus (RCDaV) is tentatively proposed. The RCDaV genome consists of an 8,987 nt positive-stranded RNA molecule, excluding a poly(A) tail, that encodes two large polyproteins. Using cleavage assays, we have identified that the RCDaV 3C protease (3Cpro) as a serine protease recognizes the conserved EPT/S cleavage site which differs from the classic Q(E)/G(S) sites cleaved by most picornaviral 3C chymotrypsin-like cysteine proteases. Therefore, we comprehensively deciphered the RCDaV genome organization and showed that the two polyproteins of RCDaV can be cleaved into 12 mature proteins. We found that seven unclassified picornaviruses also encode a 3Cpro similar to RCDaV, and use the highly conserved EPT/S as the cleavage site. The precise genome organizations of these viruses were illustrated. Moreover, RCDaV and the seven unclassified picornaviruses share high sequence identities and similar genome organizations, and cluster into a distinct clade in the order . Our study provides valuable information for the understanding of picornaviral 3Cpros, deciphers the genome organization of a few relatively obscure picornaviruses, and lays the foundation for further pathogenesis research on these viruses.