The role of HLA-B27 in the pathogenesis of spondyloarthropathies is widely recognized, but it is important to note that only a minority of HLA-B27-positive individuals are affected. Currently, the diagnosis of ankylosing spondylitis relies primarily on clinical manifestations and sacroiliac joint radiographic data. HLA-B27 can neither be used to confirm the diagnosis of ankylosing spondylitis nor to predict the patient’s prognosis, i.e., ankylosing spondylitis does not necessarily occur in those who are positive for HLA-B27; nor does it necessarily occur in those who are negative for HLA-B27. (1) HLA-B27 positivity significantly increases the chance of a correct diagnosis if the signs and symptoms suggest that the patient has more than a 50% likelihood of ankylosing spondylitis. (2) In children with inflammatory arthropathy, a positive HLA-B27 may suggest the possibility of ankylosing spondylitis. (3) HLA-B27 positivity in children of patients with ankylosing spondylitis, especially in males, suggests a greater likelihood of developing ankylosing spondylitis. (4) In patients with low back pain, HLA-B27 negativity is not conducive to the diagnosis of ankylosing spondylitis, and HLA-B27 negativity can almost always be used as a condition to rule out ankylosing spondylitis in the absence of psoriasis and inflammatory bowel disease. (5) Patients with HLA-B27-positive psoriasis or inflammatory bowel disease are likely to have concomitant ankylosing spondylitis. (6) Compared with HLA-B27-negative ankylosing spondylitis, HLA-B27-positive patients have a relatively early onset, more severe clinical symptoms, a higher incidence of systemic symptoms and peripheral arthritis (e.g., hip), more altered immunologic parameters (increased ESR, CRP, and gamma-globulin), and a higher incidence of familial clustering, bamboo-like spine, and ophthalmoplegia, i.e., HLA-B27 positive patients have more severe disease and a poorer prognosis. In contrast, HLA-B27-negative patients are more likely to have psoriasis, ulcerative colitis or Crohn’s disease. (7) HLA-B27 has no specific application in the prevention and treatment of ankylosing spondylitis, but it may have the possibility of being used for prevention, such as therapeutic abortion when HLA typing of amniotic fluid cells determines that the fetus carries the disease-causing gene, and cytotoxic HLA antibodies can be used to kill the sperm carrying the disease-causing gene and prevent the transmission of the disease-causing gene. In conclusion, HLA-B27 is a reference indicator for the diagnosis of ankylosing spondylitis and is not a basis for diagnosis. If the diagnosis of ankylosing spondylitis has been confirmed by X-ray and other imaging, HLA-B27 may not be performed again.