Background: Seronegative Spondyloarthropathies (SNSAs) are referred to a group of diseases with same clinical and genetic features. Objectives: Due to lack of data about HLA-B27 prevalence in patient with SNSA in Kashan this study was conducted to determine the status of HLA-B27 in these patients. Patients and Methods:This cross sectional study was conducted on 294 patients suffered from SNSA. Presence or absence of HLA-B27 was checked by serological method using Greener kit. Results were analyzed with SPSS-16 software using t-test and χ 2 test. Results: Of all 26.5% of subjects were HLA-B27 positive. HLA-B27 was significantly more frequent in male patients (34.1% vs. 16.5%, P < 0.001). Prevalence of HLA-B27 was 69% in ankylosing spondylitis, 31.5% in psoriatic arthritis, 18.2% in entropathic arthropathy, 17.9% in undifferentiated spondyloarthropathies, 16.1% in reactive arthritis and 12.5% in juvenile rheumatoid arthritis (P < 0.001). Conclusions: HLA-B27 has highest prevalence in ankylosing spondylitis and least prevalence in juvenile rheumatoid arthritis. HLA-B27 prevalence was in relation with gender, type of disease, family history, peripheral, and axial joints engagement.