Blood crossmatching is necessary to determine transfusion compatibility between individuals, especially for species for which blood groups have not yet been defined, such as the killer whale (Orcinus orca). This study evaluated methodology for crossmatching in killer whales from a managed care population using individuals of known lineages. Twenty killer whales were evaluated for major or minor crossmatch incompatibilities, determined by evidence of macro‐agglutination. Crossmatching incompatibilities were rarely observed, both when considering 1+ reactions as incompatibilities (~15%; 59/400 pairings with 1+ to 4+) and when omitting 1+ reactions (6%; 24/400 pairings with 2+ to 4+). A universal red blood cell donor within this population (whale T) and universal recipients of red blood cells within this population (whales E, M, O, P, R, S) were identified. Relationships were examined between the most common major crossmatch phenotypes and maternal or paternal lineages. Since these whales have not been previously transfused, the diversity of crossmatch reactions could indicate the presence and diversity of preexisting alloantibodies in killer whale plasma. This study highlights the clinical value of applying a personalized medicine approach to a managed care population.