Compassion fatigue (CF) is a topic of increasing concern because it can affect the mental wellbeing of caregivers, including those caring for or using research animals. If unaddressed, compassion fatigue may adversely impact the quality of life for personnel working with animals in research settings and may influence their decision to remain in the field. This study used a cross-sectional anonymous online questionnaire to 1) examine compassion fatigue in individuals working with research animals in Canada and the US; 2) better understand how personal and work-related factors may influence feelings of CF; 3) assess coping mechanisms used to deal with CF; and 4) determine the beneficial components of a CF support program. A questionnaire was sent to laboratory animal professionals in Canada and the US via email listserves to survey the general population of laboratory animal workers and personnel working for a large North American contract research organization (CRO). A total of 422 responses were received and analyzed (n = 154 from the general population, n = 268 from the CRO). Most participants were female (73%, 309/422); 66% (101/154) and 69% (184/268) of the general laboratory animal science respondents and the CRO respondents, respectively, reported experiencing feelings of CF. Survey participants indicated that the most influential work-related factors associated with feelings of CF were understaffing, close relationships with experimental animals, a lack of resources for coping with CF, poor relationships with superiors, and lack of training in managing CF. Respondents indicated that the most influential personal factors contributing to feelings of CF were poor mental and physical health. The most commonly reported beneficial coping mechanisms were talking to a trusted individual, getting away from work, practicing self-care strategies, increasing opportunities for physical activity, and owning or caring for companion animals.