Background HLA-B27 is a class I MHC protein that is associated with different diseases collectively called, spondyloarthropathies. It includes; 2. Ankylosing spondylitis, which causes inflammation of the bones in your spine 3. Reactive arthritis, which causes inflammation of your joints, urethra, and eyes, and in some cases lesions on your skin 4. Juvenile rheumatoid arthritis 5. Anterior Uveitis, which causes swelling and irritation in the middle layer of your eye Objectives To determine the prevalence of HLA-B27 in patients with backache at the Armed Forces Institute of Pathology in Rawalpindi. This descriptive cross-sectional study was conducted between June 2020 and Nov 2020. Material and methods Of the 243 patients, 167 males and 76 females were tested for HLA-B27 during the study period. Venous blood samples (3 mL) were collected in EDTA tubes and processed for HLA-B27 gene identification on the cell surface. A two-color flow cytometry panel was used to analyze samples using a BD FACS Canto II flow cytometer on BD FACS Diva software. The dot plot was created using an isotype control in four quadrants, and positive and negative populations of cells were identified. Results A total of 243 patients were analyzed for the presence of HLA-B27 using flow cytometry. Of the 243 patients, 167 were male and 76 were female patient.51 male patients tested positive, with a positivity rate of 30.53% among the male population and 20.98% among the general population. In contrast to the male population, 12 female patients tested positive for the HLA-B27 gene, with a positivity rate of 15.78% among the female population and 4.93% among the general population. The total positivity rate was 25.92%, with a higher prevalence in the male population included in this study. Conclusion All individuals tested for HLA B27 were included in this study. This study showed that positivity rate of HLA-B27 is more in males with a percentage of 20.98% as compared to female population (4.93%).