Canine babesiosis is suspected when there is a history of tick bites and when suggestive clinical signs are present and combined: fever, anemia, hemoglobinuria, splenomegaly. Diagnosis of babesiosis is usually performed by detection of piroplasms in Giemsa-stained blood smears from capillary blood. Intraerythrocytic pear-shaped or circular organisms or large numbers of extraerythrocytic parasites are helpful diagnostic clues. Serologic tests (IFAT, ELISA, Dot-ELISA) and methods to detect parasite DNA in blood samples after amplification with polymerase chain reaction (PCR) are available at some laboratories. Hematologic changes are non-specific (anemia, thrombocytopenia), although babesiosis should always be considered in cases of hemolytic anemia and thrombocytopenia. Blood samples may auto-agglutinate in saline and may be Coombs' positive. The major differential diagnosis is immune-mediated hemolytic anemia.

Serological testing by immunofluorescent antibody test (IFAT) or ELISA is not considered useful during acute infection or to differentiate between different Babesia species/subspecies, due to extensive cross reaction between different organisms. New, highly specific immunological tests are being developed, based on recombinant antigen technology. False negative results may occur if an acute disease onset precedes antibody development, and tests generally hardly distinguish between the different Babesia species due to antigenic cross-reactivity. However, serological testing may be very useful in sub-acute or chronic forms (parasites in low numbers or absent in blood).

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