We previously reported the first detection of simian picobirnaviruses (PBVs) by polyacrylamide gel electrophoresis in fecal specimens of two monkeys with diarrhea in China. We now report the detection of genogroup I PBVs in 48% (44/92) of the fecal specimens by reverse transcriptase PCR (RT-PCR) and amplicon sequencing using primers specific for the RNA-dependent RNA polymerase (RDRP) gene. Molecular characterization of these 44 strains demonstrated both sequence conservation and diversity among simian PBVs and among simian, porcine, and human PBVs. We further determined full-length sequences of segment 2 of the two simian PBV strains, monkey/CHN-14/2002 and monkey/CHN-49/2002, and demonstrated 52.5% to 54.2% nucleotide sequence similarity to the corresponding gene of the bovine strain RUBV and the prototype human strain 1-CHN-97 of genogroup I PBVs and an even lower similarity (38.4%) to segment 2 of the prototype human genogroup II strain 4-GA-91. Further studies are needed to investigate the epidemiology and pathogenesis of PBVs in animals and humans. P icobirnaviruses (PBVs) belong to the family Picobirnaviridae. They are small, nonenveloped viruses that are 35 nm in diameter and consist of a simple capsid with an indistinct surface structure (14). PBVs possess a bisegmented double-stranded RNA (dsRNA) genome. The large genome segment (segment 1) is 2.3 to 2.6 kb in size and encodes the capsid protein and a polypeptide of unknown function. The small genome segment (segment 2) is 1.5 to 1.9 kb and encodes the viral RNA-dependent RNA polymerase (RDRP). Based on the sequences of the small segment, PBVs are classified into genogroup I (prototype strain, 1-CHN-97) and II (prototype strain, 4-GA-91) (1, 15).PBVs were first detected in fecal specimens from humans and rats in 1988 (13, 14). Subsequently, the viruses have been detected in fecal samples from pigs (4, 11), calves (19), foals (3), rabbits (10), giant anteaters (6), birds (9), dogs and snakes (5), and monkeys (20). Since PBVs have been found in humans and animals with diarrhea and in healthy people and animals (12, 21) and in samples with or without the presence of other known enteric pathogens, the role of PBVs in gastroenteritis has not been established. PBVs have been commonly identified by the characteristic migration profiles of the bisegmented dsRNA by the use of polyacrylamide gel electrophoresis (PAGE) and silver staining. However, this method is relatively insensitive and time-consuming. The availability of a reverse transcriptase PCR (RT-PCR) greatly improved the sensitivity of PBV detection using two pairs of primers specific to segment 2 sequences (15). Amplification with these primers gave an amplicon of 201 to 207 bp for genogroup I and ϳ369 bp for genogroup II PBVs. Sequence analysis of the shorter amplicons has enabled us to perform molecular characterization and demonstrate extensive genetic diversity of PBVs within and across mammalian and avian species in geographic locations worldwide. However, the limited partial-sequence data and th...