Abstract Background Cytauxzoon felis is a life-threatening protozoan disease of cats. Identification of schizont-laden macrophages is a point-of-care diagnostic test for acute cytauxzoonosis. Hypothesis/Objectives The primary objective determined cytologic agreement between sample types to diagnose acute cytauxzoonosis. The secondary objective evaluated novices' ability to identify cytauxzoon organisms in blood films and tissue aspirates. Animals Thirty-eight cats with suspected acute cytauxzoonosis and 5 controls examined postmortem. Methods Cases were prospectively submitted and collected. Blood film, lymph node, and splenic aspirates were blindly reviewed for sample quality, presence of schizont-laden macrophages, and agreement between sample types. A subset of cases and controls were evaluated by 12 blinded novice observers to determine sensitivity and specificity for identifying organisms in various sample types. Results Acute cytauxzoonosis diagnosis was made on at least 1 sample type in 28/38 cats. Schizont-laden macrophages were seen on 33% (10/30) of blood films, 56% (19/34) lymph node aspirates, 77% (26/34) splenic aspirates. Schizont-laden macrophages were more likely seen on splenic than lymph node aspirates (McNemar's, P = .03) or blood film (McNemar's, P = <.001). Novice observers were more likely to agree with experts when identifying schizont-laden macrophages in splenic aspirates (sensitivity = 77.1%, specificity = 94.4%) versus lymph node aspirates (sensitivity = 52.8%, specificity = 96.4%) or blood films (sensitivity = 41.7%, specificity = 96.9%). Conclusions and Clinical Importance Schizont-laden macrophages are most frequently identified in spleen, even by novice observers. If the diagnosis of acute cytauxzoonosis cannot be confirmed via blood film, then splenic, followed by peripheral lymph node aspirates can be considered.