We used an indirect enzyme-linked immunosorbent assay (iELISA) and the rose bengal test (RBT) to test for anti-Brucella antibodies in moose (Alces alces gigas), muskoxen (Ovibos moschatus), and plains bison (Bison bison bison) from various game management units (GMUs) in Alaska, US, sampled from 1982 to 2010. A portion of the sera had previously been tested with the standard plate test (SPT), the buffered Brucella antigen (BBA) card test, and the card test (CARD). No antibody-positive plains bison were identified. Anti-Brucella antibodies were detected in moose (iELISA, n=4/87; RBT, n=4/87; SPT, n=4/5; BBA, n=4/4) from GMU 23 captured in 1992, 1993, and 1995 and in muskoxen (iELISA, n=4/52; RBT, n=4/52; CARD, n=4/35) from GMUs 26A and 26B captured in 2004, 2006, and 2007. A negative effect of infection on the health of individuals of these species is probable. The presence of antibody-positive animals from 1992 to 2007 suggests presence of brucellae over time. The antibody-positive animals were found in northern Alaska, an area with a historically higher prevalence of Brucella-positive caribou, and a spillover of Brucella suis biovar 4 from caribou may have occurred. Brucella suis biovar 4 causes human brucellosis, and transmission from consumption of moose and muskoxen is possible.