Primary immune-mediated hemolytic anemia (IMHA) is thought to be uncommon in cats; secondary IMHA, induced by neoplasia or infectious agents, is considered common. Neither mechanism can be detected reliably using standard Coombs’ testing to identify anti-erythrocyte antibodies. Recent studies suggest that primary IMHA may be more common in cats than previous thought. The purpose of this prospective study is to identify a subset of cats with primary IMHA and to develop more sensitive tests for detecting autoantibodies in cats with either primary or secondary IMHA. Whole blood and serum samples from 7 healthy client-owned cats and 43 client-owned cats presenting for acute anemia will be analyzed using polymerase-chain reaction assays for infectious agents known or suspected to cause secondary IMHA (Mycoplasma haemofelis, ‘Candidatus M. haemominutum,’ feline leukemia virus (FeLV), Ehrlichia canis-like organisms, Anaplasma phagocytophilum, Cytauxzoon felis and Bartonella spp) and evaluated for anti-erythrocyte antibodies using the Coombs’ test. Flow cytometry and an ELISA essay, two previously untested techniques in cats, will be validated and compared to the Coombs’ test. The recognition of primary IMHA in cats and the use of more sensitive techniques to characterize feline IMHA may lead to new diagnostic and therapeutic approaches to the disease.