A number of serological tests were compared for the detection of antibodies to Brucella abortus in bison (Bison bison). The performance of the fluorescence polarization assay (FPA) in both the preliminary evaluation and a subsequent blind validation indicated that this test was the most suitable for serological diagnosis of brucellosis in bison. The sensitivity and specificity in the preliminary evaluation were 92.1% and 99.4%, respectively. The sensitivity and specificity in a subsequent blind study were 96.3% and 97.6%, respectively. In a double blind study conducted on bison vaccinated with B. abortus strain 19, the data suggests that the FPA can differentiate bison infected with B. abortus from bison vaccinated with B. abortus strain 19. Both the indirect immunoassay (IELISA) and the competitive immunoassay (CELISA) performed nearly as well as the FPA. The buffered antigen plate agglutination test (BPAT) and the complement fixation test (CFT) did not perform as well as the FPA, CELISA or the IELISA in both studies. The FPA is a homogeneous assay eliminating the washing steps and reducing incubation to minutes rather than hours saving on time, equipment, materials, reagents and cost. These attributes, together, with its excellent sensitivity and specificity make the FPA an attractive test for the detection of serum antibodies to Brucella abortus in bison.