Objective:To construct a scientific molecular typing criteria for coxsackievirus B5 (CVB5) according to the basic principles of molecular typing for enteroviruses (based on the cut-off value of 15%-25% difference in the entire nucleotide sequence of VP1 region). Methods:The entire VP1 coding regions of all 69 CVB5 strains isolated from mainland of China from 2001 to 2021 were amplified by reverse transcription-polymerase chain reaction using the Chinese Hand, Foot, and Mouth Disease (HFMD) Laboratory Network Platform. The nucleotide sequencing and analyses were also performed. The 742 complete sequences of VP1 coding region (849 nucleotides) of CVB5 in GenBank by the end of February 16, 2022 were downloaded and filtered. The phylogenetic tree with maximum likelihood method for the study of CVB5 genotyping criteria was constructed using MEGA 7.0 software with the bootstrap value of 1000.Results:Nucleotide and amino acids sequence similarity between 69 CVB5 isolates from the Chinese mainland and CVB5 prototype strain (Faulkner strain) ranged from 77.3% to 81.6% and 93.2% to 95.3%, respectively. The phylogenetic tree showed that CVB5 worldwide could be divided into 4 different branches. The average differences in nucleotides and amino acids within the branch were 7.3%-13.4% and 1.9%-3.3%, respectively. The average differences s in nucleotides and amino acids between branches were 17.0%-22.3% and 4.2%-4.9%, respectively. According to the above molecular genotyping criteria, CVB5 worldwide could be divided into four genotypes: A, B, C and D. Genotypes A and C might have disappeared during the evolution and were no longer prevalent. Genotypes B and D of CVB5 presented a co-epidemic trend worldwide. The predominant genotype of CVB5 in the Chinese mainland was genotype D with a small proportion of genotype B.Conclusions:In this study, the criteria for molecular typing of CVB5 was established and the CVB5 strains worldwide were classified into four genotypes accordingly. This is of important value for further understanding of the genetic variation and transition of CVB5 as well as for molecular traceability.