The association of human leukocyte antigen (HLA)-B27 with ankylosing spondylitis (AS) has been known for over 38. years. However, it is not the only gene associated with AS. The aim of this study was to confirm the association of HLA markers around HLA-DPA1/DPB1 region with AS in HLA-B27 positive populations. Five SNPs (rs422544, rs6914849, rs92777535, rs3128968 and rs2295119) from the HLA-DPA1/DPB1 region were genotyped in 340 individuals HLA-B27-positive from Portugal (137 AS patients and 203 healthy controls). Characterizations of HLA-DPA1/DPB1 alleles were also performed. rs422544 revealed a significant association with AS (. P<. 0.05) and sliding windows (SW) analysis showed association of some groups of adjacent SNPs within HLA-DPA1/DPB1 region with AS (. P<. 0.05). We also found association of the HLA-DPA1*01:03 allele with AS (. P<. 0.05). This is the first study that confirms the association of HLA markers and haplotypes around HLA-DPA1 and HLA-DPB1 with AS.