Background: Most recently, murine kobuvirus (MuKV), a novel recognized member of the family Picornaviridae, has been initially identified from fecal samples of Rattus norvegicus in China. The circulation of MuKV from other murine rodent species is not fully understood, prompted us to investigate the prevalence and genetic characterization of MuKV in Rattus losea, Rattus tanezumi and Rattus norvegicus in Southern China. Results: Between 2015 and 2017, a total of 243 fecal samples from three murine rodent species were screened for the presence of murine kobuvirus (MuKV) from three cities in Southern China, with an overall prevalence of 23.0% (56/243). Phylogenetic analysis based on the complete VP1 gene suggested our sequences were genetically closely related to each other and the Chinese strains. Three complete polyprotein nucleotide sequences of MuKV and the genome organization were acquired in the present study. Phylogenetic analyses also showed our sequences belong to the members of genus Kobuvirus within the genotype of Achivirus A. Conclusion: The present study indicated that MuKV is very common in murine rodent populations.