Rotaviruses (RVs) account for severe diarrhea in children and young animals globally. In the current study, the fecal samples of diarrheic calves from a beef farm in Inner Mongolia were screened for RVA by ELISA and RT-PCR, followed by culture of three positive RVA samples in the MA-104 cell line. After 10 blind passages, cytopathic effects (CPE) appeared as detachment, granulation, and clustering of the inoculated cells. The virus isolates were identified by RT-PCR (VP6 gene RVA) and ESI-LC-MS/MS for whole protein sequencing. The protein sequences demonstrated the presence of two strains from species A rotavirus and one RVB strain; RVA/Cow-tc/CHN/35333/2019/G6P was mixed with one RVB strain (RVB/Cow-tc/CHN/35334/2019/G5P) in two samples, and RVA/Cow-tc/CHN/10927/2019/G8P was found in one sample. They are of genotype constellations (G6-P-I2-R2-C2-M2-A3-N2-T6-E2-H3), (G8-P-I5-R1-C1- M2-A1-N1-T1-E1-H1), and (G5-P-I3-R5-C5-A5-N4-H5), respectively. Besides, phylogenetic analysis of the obtained sequences demonstrated viral evolution.Copyright © 2021. Published by Elsevier B.V.