Bufavirus (BuV) was initially discovered in fecal samples from children with acute diarrhea. In this study, we determined the prevalence, distribution, and genotype(s) of BuV in Thailand. A total of 1,495 diarrheal and 741 non-diarrheal stool specimens were collected and analyzed. A portion of the NS1 gene of BuV was amplified by nested RT-PCR. Phylogenetic analysis was performed to classify the BuV strains found. We detected bufavirus (BuV) in diarrheal (4/1495; 0.27%) but not in non-diarrheal specimens (0/726). All four strains belonged to BuV genotype 1. BuV could be detected in adults and children, but its role in causing acute diarrhea remains unclear.