The perceptions, knowledge, and gaps in the knowledge of Ohio bovine veterinarians about antibiotic resistance (ABR) issues were systematically determined. In-depth interviews of 10 bovine practitioners in Ohio were completed to inform the development of a survey that was subsequently distributed to all Ohio members of the American Association of Bovine Practitioners and the bovine-specialized members of the Ohio Veterinary Medical Association (n = 174). The 43 respondents (26% response rate) included practitioners with a cross-section of clinical experience and sizes of dairy herds served. Respondents' knowledge regarding selection pressures that cause bacteria to acquire resistance was positively correlated with their knowledge of transmission routes of ABR organisms to dairy cows (r = 0.60) and potential consequences of ABR with respect to animal health (r = 0.52). Veterinarians believed that antibiotics are used by producers to treat a variety of illnesses and that specific antibiotics (e.g., gentamicin) are used without veterinary consultation. More than 75% of the veterinarians thought that one-on-one meetings and handouts containing good management practices, diagnosis descriptions, and appropriate dosages for antibiotics would be effective ways to educate their clients about ABR. However, only 23% of veterinarians reported that they consistently provide treatment protocols for antibiotic use. By first assessing the information needs of the audience, these results indicated that, rather than expending resources to develop educational materials directed at improving bovine practitioner knowledge of the subject, communication and outreach efforts that encourage and facilitate information flow from veterinarians to dairy producers may be more effective tools to affect prudent antibiotic use on dairy farms.