The aim of this study was to investigate the association of the B27 subtypes with ankylosing spondylitis (AS) in the Wuhan population of China. We selected 317 HLA-B27-positive individuals (145 controls and 172 patients with ankylosing spondylitis). The B27 subtypes were characterized using a PCR-SSP method. Six B27 subtypes were determined: B*2702, 03, 04, 05, 06 and B*13. HLA-B*2704 and HLA-B*2705 were the two high frequency genotypes in controls and patients. Compared with the controls, the AS patients had high frequency of B*2704 (patients 69.2% vs. controls 53.8%) and low frequency of B*2705 (patients 23.8% vs. controls 33.1%). B*2703 was detected in 10 (5.8%) patients and in 13 (8.9%) controls. B*2702, 06 and B*2713 were relatively rare. Our results show that the allele conferring risk to AS in the Wuhan population of China was B*2704 and B*2705. B*2704 is strongly associated with AS.