The purpose of this study was to survey the seroprevalence of infection with the agents of production-limiting diseases in dairy cattle in New Brunswick, Nova Scotia, and Prince Edward Island. In 30 randomly selected herds per province, 30 cattle per herd were randomly selected and tested for antibodies to bovine leukemia virus (BLV) and Mycobacterium avium subspecies paratuberculosis (M. paratuberculosis), while 5 unvaccinated cattle over 6 months of age were tested for antibodies to bovine viral diarrhea virus (BVDV). For BLV, 20.8% (15.8% to 27.0%) of cows were positive, and 70.0% (60.3% to 79.7%) of herds had at least one positive cow. In BLV-positive herds, the average BLV prevalence was 30.9% (24.8% to 37.2%). For M. paratuberculosis, 2.6% (1.8% to 3.9%) of cows were positive, and 16.7% (8.8% to 24.5%) of herds had at least 2 M. paratuberculosis-positive cows. In M. paratuberculosis-positive herds, the average M. paratuberculosis prevalence was 8.5% (6.9% to 10.1%). For BVDV, 46.1% (35.5% to 56.7%) of herds had at least 1 BVDV-positive animal with a titer greater than or equal to 1:64.