Nonpathogenic and opportunistic bacteria are increasingly recognized as reservoirs of antibiotic resistance genes. However, nothing is known about the mechanisms of antibiotic resistance in such bacteria isolated from the udders of healthy animals. In this study, 150 Gram-negative strains isolated from milk samples of healthy dairy cows were screened for the presence of a large pool of antibiotic resistance markers. Strains carrying β-lactamase-resistance genes, including SHV-1, SHV-11, SHV-27, TEM-1, OXY-1, CTX-M-2 and class 1 integrons, were detected. Our findings give the first evidence that nonpathogenic and opportunistic bacteria carrying antibiotic resistance genes can asymptomatically invade healthy udders and suggest that they may play a role in the dissemination of antibiotic resistance genes to the other udder pathogens.