Protocols for crossmatch reactions vary in veterinary medicine, particularly regarding the use of recipient serum vs plasma. Sources suggest that major crossmatch results might differ when recipient plasma is used instead of serum, but there are conflicting reports as to the exact effects on the results. The aim of this study was to determine the frequency and degree of discrepancy, if any, between canine major crossmatch reactions using serum versus EDTA plasma, performed via a standard tube method. One hundred duplicate canine major crossmatch reactions were performed with both serum and EDTA plasma from 100 different "recipient" dogs against erythrocytes from a single, healthy "donor." Decreasing concentrations of a rabbit anti-dog erythrocyte antibody were added to generate strong positive, weak positive, and negative results for each crossmatch reaction. Crossmatch results were followed through the following phases: immediate spin, cold, warm, albumin, and Coombs. Semi-quantitative results were compared between reactions using serum vs EDTA plasma. Weak positive, major crossmatch reactions were significantly more likely to demonstrate stronger agglutination in EDTA plasma compared with serum in the immediate spin phase, cold phase, warm phase, and albumin phase (P<0.001). There was no statistically significant difference between serum and EDTA plasma results in the Coombs' phase (P=0.313). In this experimental setting, EDTA plasma and serum were both deemed acceptable for use in canine major crossmatch reactions. EDTA plasma might be preferable to detect weak agglutination with more sensitivity and reduce recipient blood volumes needed to complete the crossmatch reaction.