Objective To study the molecular polymorphism and the distribution of HLA-B27 subtypes in southern Chinese Han patients with Ankylosing Spondylitis and healthy controls.Methods A total of 46 samples form southern Chinese Han patients with AS and 80 non-related blood samples from healthy peripheral blood stem cell donors with B27-positive identified by rSSO Lumminex flow array assay were subjected to sequencing analysis of exon 2 ,3 and 4 of HLA-B gene by the sequence-based typing,the purified products of sequencing reaction were electrophoresed on ABI 3730 DNA sequencer and the designation of HLA-B27 allele was accomplished using the Assign3.5 software. The ambiguities and the detected "rare" alleles were confirmed using the PCR-SSP commercial kit. Results In the 46 B27-positive patients with the diagnosis of AS,four alleles,namely B2704,B2705,B2707 and B2724 were determined. The frequencies for these four alleles were 82.98%(39/47),12.77%(6/47),2.13%(1/47) and 2.13%(1/47),respectively. In the 80 B27-positive control individuals,seven B27 related alleles were identified. The frequency for the two dominant subtype B2704 and B2705 were 57.32%(47/82) and 26.83%(22/82),respectively. Both the B2706 and B2707 were observed 5 times with a frequency of 6.10%(5/82),three alleles B2703,B2715 and B2724 were detected only once with a frequency of 1.22%(1/82).Conclusion Our study shows that HLA-B2704 and B2705 were the predominant subtypes in normal healthy controls,however,B2704 was the predominant subtype for the AS group in southern Chinese Han patients.