BACKGROUND: HLA-B27 can effect clinical presentation and course of ankylosing spondylitis. Different detection techniques of HLA-B27 are available with variable sensitivities and specificities. OBJECTIVE: To compare serologic and molecular diagnostic techniques of detecting HLA-B27 status and to correlate it with some clinical variables among ankylosing spondylitis patients. PATIENTS AND METHODS: A cross-sectional study was conducted on 83 Iraqi patients with ankylosing spondylitis. Clinical and laboratory evaluations were reported. HLA-B27 status was determined in all patients by real-time PCR using HLA-B27 RealFast™ kit; ELISA method was used as well to detect soluble serum HLA-B27 antigens using Human Leukocyte Antigen® kit. RESULTS: The mean age of patients ± SD was (38.4±9.8) years. Male to female ratio was 9:1. Disease onset occurred <30 years in 78% of cases. All (100%) cases had lower back pain, 44 (54%) patients had enthesitis, 14 (16.9%) had peripheral arthritis, 12 (14.5%) had eye involvement, whil