Technical Abstract: Numerous species of wild mammals are susceptible to Mycobacterium bovis, a cause of bovine tuberculosis (TB). Eurasian badgers, white-tailed deer, brushtail possums, and wild boar are implicated in the maintenance of wildlife reservoirs of M. bovis infection in different countries, fueling bovine TB in domestic livestock. Rapid and accurate assays that can detect TB in a variety of species under field conditions are needed to improve wildlife surveillance efforts worldwide. Using two membrane-based serological assays, a lateral-flow rapid test (RT) and MultiAntigen Print ImmunoAssay (MAPIA), antibody responses during M. bovis infection in the four natural hosts of were characterized. Predominantly recognized antigens were identified and heterogeneity of antibody responses in each species was shown. The utility of RT as a single serologic assay for detection of bovine TB in badgers, deer, possums, and wild boar was demonstrated. The diagnostic accuracy of RT varied from 72% in possums to 97% in deer. Use of whole blood from white-tailed deer demonstrated the potential of this approach with RT for an expedited detection of M. bovis infection in wildlife populations under field conditions. A close association between antibody responses and the presence of gross TB lesions found in wild boar suggested that RT could predominantly identify animals with most advanced disease, which have the greatest potential of shedding M. bovis. Therefore, serologic assays may provide useful screening tools for controlling bovine TB in multi-species wildlife populations.