Abstract: In order to understand the infection and molecular characteristics of bovine Kobuvirus（BKV） in dairy and beef cattle in Guangxi region, in this experiment, the RT-PCR method was used to detect BKV and other enteroviruses in 177 cow feces and 27 beef feces（a total of 204 feces, including 82 diarrheal feces and 122 normal feces） collected from Guangxi region. The co-infection of BKV with other enteroviruses, as well as the infection of BKV in different locations and seasons, was analyzed; and the nucleotide similarity and genetic evolution were analyzed according to the 3D sequences of BKV in Guangxi region and the 3D sequences of 30 Kobuviruses at home and abroad. The results showed that the infection rate of BKV in Guangxi region was 2.94%（6/204）, all of which were detected in diarrheal feces; among them, the infection rates of dairy cows and beef cattle were 2.26%（4/177） and 7.41%（2/27）, respectively. The sequences of the six strains were named BKV-GXGG01（Guigang strain）, BKV-GXHC01（Hechi strain）, BKV-GXHC02（Hechi strain）, BKV-GXNN01（Nanning strain）, BKV-GXNN02（Nanning strain） and BKV-GXNN03（Nanning strain）. BKV had mixed infection with Bovine viral diarrhea virus, Bovine enterovirus and Bovine astrovirus, with an infection rate of 66.67%（4/6） and a single infection rate of 33.33%（2/6）. The nucleotide similarity between the 3D sequences of the six BKV strains in Guangxi region was 93.5%-100%; the nucleotide similarity with the domestic and foreign Aichivirus B reference strains was 89.9%-95.1%, and the nucleotide similarity with other Kobuvirus reference strains was 35.9%-75.2%. The six strains in Guangxi region all belonged to Aichivirus B class in genetic evolution analysis, the strain BKV-GXNN01 belonged to the same branch as the Japanese strain K-35, Henan strain B4, Inner Mongolia strain H6 and H18, Shandong strain C2, Heilongjiang strain SC7-5, and Liaoning strain H9; and the remaining 5 strains BKV-GXGG01, BKV-GXHC01, BKV-GXHC02, BKV-GXNN02, BKV-GXNN03 belonged to the same branch as Beijing strain B1213, Thailand strain CMB02, and Japanese strain U-1. The results suggested that BKV was presented in diarrhea cases in dairy and beef cattle in Guangxi region, which was speculated that BKV might be related to bovine diarrhea.