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Objectives: Human leukocyte antigen (HLA) B27 is a susceptibility allele of ankylosing spondylitis (AS), and HLA-B27 antigen typing is an important indicator for clinical diagnosis of AS, but current typing methods such as sequence specific primer polymerase chain reaction (PCR-SSP) still possess limitation. Therefore, this study aims to analyze the correlation between B27 subtypes and susceptibility to AS in Hunan Province by applying high-resolution polymerase chain reaction-sequence-based typing (PCR-SBT)., Methods: Peripheral blood of 116 patients with suspected AS (suspected AS group) and 121 healthy volunteers (control group) admitted to the Second Xiangya Hospital from January 2020 to December 2020 were collected for HLA-B genotyping by PCR-SBT. Among the patients in the suspected AS group, 23 patients were finally diagnosed with AS (confirmed AS group), and the remaining 93 undiagnosed patients served as the non-confirmed AS group. PCR-SBT and PCR-SSP were used to detect HLA-B27 typing in 116 patients with suspected AS, and the results of the 2 methods were compared., Results: The HLA-B27 allele frequency in the suspected AS group was significantly higher than that in the control group [11.63% vs 2.48%; P <0.001, odds ratio ( OR )=5.18, 95% confidence interval ( CI ) 2.097 to 12.795]. B*27:04, B*27:05, B*27:06, and B*27:07 were detected in the suspected AS group and the control group. The frequency of the B*27:04 allele in the suspected AS group was significantly higher than that in the control group (9.48% vs 1.24%; P <0.001, OR =8.346, 95% CI 2.463 to 28.282). The positive rate of B27 in the suspected AS group and the confirmed AS group (B27+/+ and B27+/-) was significantly higher than that in the control group ( χ 2 =16.579, P <0.001; χ 2 =94.582, P <0.001, respectively). Among the confirmed AS group, 21 were HLA-B27 carriers, and the B27 positive rate in the confirmed AS group was 91.3%. PCR-SBT could achieve high resolution typing of the HLA-B gene locus, with higher sensitivity, specificity, positive predictive value, negative predictive value, and accuracy than PCR-SSP., Conclusions: PCR-SBT typing analysis shows a strong correlation between HLA-B * 27:04 and AS in Hunan province. The PCR-SBT method can be used as the preferred option for the auxiliary diagnosis of clinical AS.