The HLA-B35 serotype represents a group of antigens detectable by IEF, cytotoxic T cells, and by sequencing analysis. Four isotypes and eight alleles have been thus far reported. We have determined the relative frequencies of these B35 subtypes in a group of 203 unrelated people. Dot blot hybridization of PCR amplified products was performed using 23 sequence-specific oligo probes designed based on the EMBL HLA class I sequence database. The amplification was achieved by a pair of group-specific primers, producing approximately 600 bp fragments. By hybridization pattern analysis, we found that four alleles represent over 95% of the B35+ population, with relative frequency of 48.2% for B*3501, 23.7% for B*3502, 15.2% for B*3503, and 8.0% for B*3508. We also identified 3 individuals with B*3504 and one with B*3505, and seven samples with new patterns. B*3501 and B*3503 exactly correlated with the most common isotype B35.3, B*3502 and B*3504 with B35.2, B*3508 may be the B35.1 IEF isotype. The B*3505 was identified from an individual with B35 IEF variant form. Our study shows that the B35 antigen has a wide distribution of alleles, and that many more B35-related alleles may yet to be uncovered.