Blood donation is suggested to increase the risk of Restless Legs Syndrome (RLS). This study aims to assess the prevalence of RLS in Spanish blood donors and determined its potential correlation with iron metabolism parameters. Prospective cohort study of 129 blood donors (54.3% men, 39.44 years ± 11.0) that underwent a physical examination, blood analysis (hemoglobin, ferritin, transferrin saturation index and soluble transferrin receptor) and a RLS screening questionnaire followed by a prospective follow-up study including a clinical phone interview. A multivariate logistic regression model was performed to examine the association between RLS and other variables. Eighty-four (65.1%) participants were repeat blood donors (mean of 2.11 donations/year) at inclusion and 61 (47.4%) at follow-up (mean of 2.09 donations/year). Non-anemic iron deficiency (ferritin < 50 µg/L) was high in women p < 0.001 and in repeat donors (p = 0.003). The prevalence of RLS was 14.1% at inclusion increasing prospectively (19.5%; p = 0.065). On multivariate analysis, gender was the only variable significantly associated with a RLS diagnosis, being higher in women (OR 5.1; 95% CI 1.71-15.3; p = 0.003). Despite the high prevalence of non-anemic iron deficiency there was no association between ferritin, soluble transferrin receptor concentration values and RLS diagnosis. Gender was associated with RLS diagnosis regardless of other variables.