Current immuno-diagnostic tests for the detection of Mycobacterium bovis infection in cattle rely on the use of tuberculin PPD as antigens. However, the use of a cattle vaccine is effectively prohibited because BCG, the only potentially available cattle TB vaccine, compromises the current tuberculin test. The main objective of this study was to identify specific antigens, which could increase the test sensitivity to levels achieved with tuberculin. Our approach utilized the availability of the genome sequences of Mycobactereium tuberculosis and BCG by applying principles of comparative genomics to the identification of species-specific antigens. Eight open-reading frames (Rv3871 to Rv3878) encoding for putative antigens in the RD1 region of the M. tuberculosis genome, which is deleted in all strains of BCG, were selected and screened in the form of pools of synthetic peptides for immunological reactivity (antigen induced proliferation and IFN-γ secretion) with peripheral blood mononuclear cells from cattle experimentally infected with M. bovis. Our results confirm the immunodominant role of two RD1 region products, CFP-10 (Rv3874) and ESAT-6 (Rv3875). In addition, we were able to identify 3 more antigens (Rv3871, Rv3872 and Rv3873), which induced immunological reactivity in PBMC from more than 50% M. bovis of infected cattle.