Background: Flow cytometry has been used to detect anti‐red blood cell (RBC) antibodies in dogs with immune‐mediated hemolytic anemia (IMHA), but the prevalence of anti‐RBC antibodies in anemic and nonanemic dogs with a variety of different diseases has not been assessed previously. Hypothesis: We hypothesized that anti‐RBC antibodies would be more common in anemic dogs and in dogs with immune‐mediated disorders and cancer. Animals: Blood samples from 292 dogs were analyzed prospectively by flow cytometry for anti‐RBC antibodies. Methods: Blood samples from 147 anemic and 145 nonanemic dogs were evaluated by flow cytometry to detect surface‐bound immunoglobulin (Ig) G and IgM antibodies on RBC. Disease associations with RBC antibodies were determined, as was the correlation between disease status and the percentage of Ig+ RBC. The specificity and sensitivity of flow cytometry and clinical variables for the diagnosis of IMHA were compared by Bayesian analysis. Results: Anemic dogs were significantly more likely to be positive for anti‐RBC antibodies (IgG, IgM, or both) than nonanemic dogs. Anemic dogs also had significantly higher percentages of Ig+ RBC than nonanemic dogs, whereas dogs with IMHA had significantly higher percentages of Ig+ RBC than dogs with all other diseases. Dogs with IMHA, infectious diseases, and immune‐mediated thrombocytopenia were significantly more likely to have anti‐RBC antibodies than dogs with other medical or surgical diseases. Conclusions: Anemic dogs with immune‐mediated diseases and infectious diseases were at the highest risk for the development of anti‐RBC antibodies, and flow cytometry for the detection of IgG on RBC was highly sensitive and specific for the diagnosis of IMHA.