To assess HLA-B27 influence on the clinical phenotype of Ankylosing Spondylitis (AS) patients. An observational, cross-sectional and descriptive study of AS patients from the Spanish REGISPONSER database was performed. Demographic, clinical, disease activity (Bath Ankylosing Spondylitis Disease Activity Index (BASDAI), Bath Ankylosing Spondylitis Functional Index (BASFI), erythrocyte sedimentation rate (ESR), and C-reactive protein (CRP)), and radiographic data (Bath Ankylosing Spondylitis Radiology Index (BASRI) score) were compared regarding HLA-B27 status. A univariate and multivariate analysis was performed to identify variables independently related to the presence of HLA-B27. Data from 1235 patients (74.8% male) were analyzed; 1029 were HLA-B27 positive (83%). HLA-B27-positive patients showed higher family aggregation and an earlier onset of disease compared with those who were HLA-B27 negative. HLA-B27-negative patients presented statistically higher BASDAI and BASFI scores and higher prevalence of arthritis, dactylitis, and extra-articular manifestations (psoriasis and inflammatory bowel disease (IBD)) but not anytime uveitis compared with those who were HLA-B27 positive. In the multivariate analysis, family history (odds ratio (OR) 2.10, 95% confidence interval (CI) 1.27-3.49), younger age at diagnosis (OR 0.97, 95% CI 0.96-0.98), presence of peripheral arthritis (OR 0.53, 95% CI 0.32-0.89), dactylitis (OR 0.16, 95% CI 0.05-0.56), psoriasis (OR 0.45, 95% CI 0.26-0.78), and IBD (OR 0.22, 95% CI 0.12-0.40) were the main variables independently related to the presence or not of HLA-B27. In Caucasian AS patients, the presence of HLA-B27 is related to an earlier disease onset and higher family aggregation. Absence of HLA-B27 is related to a higher frequency of peripheral arthritis, dactylitis, and extra-articular manifestations. Being HLAB27 positive is not related to a higher burden of disease or anytime uveitis