The differences in amino acid residues of HLA-B27 subtypes are minor, but may play role in pathogenesis of ankylosing spondylitis (AS). Aim of this study was to investigate of frequency of B27 subtypes in Croatian AS patients and B27 positive healthy controls. Group of 50 AS patients and 38 B27 positive controls were typed for B27 subtypes by PCR-SSP method. In the group of AS patients we found four subtypes: B*2705 (83.0%), B*2702 (13.2%), while remaining two alleles B*2701 and B*2704 had one individual each. In the group of B27+ controls we also observed B*2705 (76.3%) as most frequent allele while frequency of B*2702 was 21.1%. No significant evidence for association between AS and a particular HLA-B27 subtype in the Croatian population were found.