Human leucocyte antigen-B*27 is strongly associated with a number of rheumatic diseases, including ankylosing spondylitis and reactive arthritis. Targeted detection of the B*27 group by molecular methods is hampered by the extreme heterogeneity of the serological B*27 group. Here, we describe a simple, rapid sequence-specific primer-based method for detection of all 28 B*27 alleles defined to date. The method involves an initial screening with two sequence-specific polymerase chain reactions (PCRs), which has to be followed by two additional PCR amplifications in samples carrying a few rare subtypes of B*27, B*4202 or B*7301. The described protocol should be useful for laboratories involved in diagnostics and research of rheumatoid diseases.