Brucellosis is a zoonotic disease which affects humans, farm animals and wildlife as well. In Pakistan, the status of human and farm animal brucellosis is documented but the information about brucellosis in wildlife is lacking. To access the seroprevalence of Brucella antibodies in avian (turkeys, peafowl, guineafowl, mallard ducks and Indian blue rock pigeon) and selected wild species (reptiles and amphibians), a total of 117 serum samples (reptile = 34, amphibian = 04, avian n = 79) were collected from Karachi and Pattoki regions of Pakistan. Serum samples were screened for Brucella antibodies using Rose Bengal plate test (RBPT) and11.1% serum samples were found positive. Overall, 2.5% birds, 29.4% reptiles and 25% amphibians were seropositive for Brucella antibodies. This is first report of detection of Brucella antibodies in reptiles and amphibians in Pakistan.