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The prevalence of HLA-B27 in the general population and in axial spondyloarthritis (axSpA) patients in Saudi Arabia is unknown. The aim of this study was to evaluate the prevalence of HLA-B27 in these two populations and describe the delay in diagnosis of axSpA patients. The prevalence of HLA-B27 in the general population was evaluated using cord blood and healthy organ transplant donor databases. Data from patients with axSpA were collected retrospectively from five centers. Ankylosing spondylitis (AS) was diagnosed based on a positive X-ray, as evaluated by two independent readers. Patients with inflammatory bowel disease and psoriasis were excluded. A total of 134 axSpA patients were included, of whom 107 (79.9%) had AS, and most (67.2%) were males. HLA-B27 was positive in 60.4, 69, and 25.9% of patients with axSpA, AS, and non-radiographic axSpA (nr-axSpA), respectively. The median and interquartile range (IQR) ages at symptom onset and disease diagnosis were 26 (20–33) and 30 (25–38) years, respectively. The median delay to diagnosis was 3 (1–6) years. There was a negative correlation between the time of onset of symptoms and the delay in diagnosis (r = −0.587). Male gender and HLA-B27 positivity were associated with a younger age at symptom onset/diagnosis (p