Abstract Enterovirus B83 (EV-B83) is a new member of the enterovirus B group. Currently, there are only two full-length genomic sequences of EV-B83 in the GenBank database and few VP1 region sequences. The aetiology and epidemiology of EV-B83 is unclear. 24 stool specimens were collected from twelve AFP patients and 298 stool specimens were collected from 298 healthy children in support of polio eradication activities in Tibet in 1999. Two polioviruses (isolated by L20B cell) and one non-polio enterovirus (isolated by RD cell) were isolated from AFP patients and nine polioviruses (isolated by L20B cell) and 90 non-polio enteroviruses (isolated by RD cell) were isolated from health children. Through molecular typing, we confirmed that the six of non-polio enteroviruses belong to EV-B83. The sequence similarity between the VP1 region of the Tibet isolates and that of the EV-B83 prototype strain was 80%. The maximum-likelihood phylogenetic tree of the partial VP1 region in EV-B83 demonstrated that EV-B83 formed four genotypes globally during the evolution process. The six Tibet EV-B83 strains formed the D genotype alone. Recombination analysis of Tibet EV-B83 showed that CV-B4, CV-A9, EV-B80, and EV-B106 may act as recombinant donors in multiple regions. The serum neutralization test showed that the antibody-positive rate was 58.8% and GMT was 1:19.70, which was higher than the previously reported results of EV-B106 and EV-B80. Temperature sensitivity test results showed that the six Tibet EV-B83 strains were temperature-insensitive with stronger virulence and potential infectivity, which was consistent with the results of the serum neutralization test. This study enriched the genome-wide sequence, epidemiological characteristics, and provided basic data for the follow-up study of EV-B83.