Since an indirect fluorescence immunoassay (IFA) for the detection of specific antibodies againstBabesia divergensin human sera is not commercially available, an in-house preparedB. divergensIFA for the examination of bovine sera was established for serological studies in humans. To determine whether the described IFA is appropriate for such studies, 2B. divergensantigens (of human or bovine origin) were tested using serum samples obtained from febrile human patients with a history of ‘tick bite’. Sera from other species of animals infected withB. divergens,BabesiaEU1,B. microtiorB. caniswere also included for comparative purposes. All serum samples were also tested using a commercially available IFA for the detection of antibodies againstB. microti, and the results compared with those obtained using blood smear and molecular techniques. This study showed that the evaluation and standardization of aB. divergensIFA for testing human sera is critical and that differentB. divergensantigens provide different end-point titres of antibodies, leading to false negative or positive results. Serological cross-reactivity betweenB. divergensandBabesiaEU1 needs to be taken into account when interpreting IFA results.