© 2018 Elsevier B.V. Besnoitia besnoiti is the causative agent of bovine besnoitiosis, a chronic and debilitating disease of cattle that recently re-emerged and seems to be spreading in Europe. A cross-sectional serological study was carried out in different cattle herds in Catalonia, north-eastern Spain, to determine the seroprevalence of B. besnoiti in the region. A total of 791 serum samples (beef cattle n = 338, dairy cattle n = 291; bullfighting cattle n = 162) were tested. Sera were first screened for antibodies against Besnoitia using a commercial Enzyme-Linked Immunosorbent Assay (ELISA) applying a cut-off that was lower than that recommended by the manufacturer in order to reach highest sensitivity. Sera above the chosen cut-off of 15% positivity (PP) were further tested by the Indirect Fluorescent Antibody Test (IFAT) and respectively positive results were confirmed by a B. besnoiti tachyzoite-based immunoblot. A total of 504/791 (63.7%) sera showed ELISA values above the selected cut-off, and 91 of these samples also yielded positive results in IFAT (cut-off titre 1:200). By immunoblot, a positive result was obtained in 93.4% (85 out of the 91) of the IFAT-positive samples. Interestingly, all confirmed Besnoitia-seropositive cases corresponded exclusively to beef cattle from the Pyrenees area, resulting in a prevalence of 25.1% (85/338) at the animal level and of 46% (36/78) at the herd level in this cattle group. No specific antibodies against Besnoitia could be detected in dairy and bullfighting cattle. The obtained results suggested that Besnoitia infections are present in Catalonia, consequently, diagnosis of this parasitic infection should be included in the sanitary control and before trading and movement of animals.