Modern molecular methods use VP1 coding region as a target for RT-PCR assays followed by sequencing, in order to identify new untyped enteroviruses' strains. In the present study, two different genomic portions of VP1 and the full length of 2A coding region of 53 clinical isolates, mostly belonging to HEV-B species, were amplified and sequenced. Nucleotide analysis of the produced sequences revealed that the values that define an unknown strains serotype vary according to the serotype and the specific part of VP1, which is investigated. The correlation, however, with the serotype was affirmed in both VP1 portions that were studied, as well as in the first 20 bases of 2A region. In the rest of 2A, no correlation with the serotype and disruption of monophyly was observed. Phylogenetic analysis of the same sequences confirmed, in most cases, the results of the nucleotide analysis.