Anticoagulants are employed to prevent clotting and preserve cellular morphology for clinical pathology tests. Lithium heparin (LH) is the most frequently used anticoagulant in chelonians; however, dipotassium ethylenediaminetetraacetic acid (EDTA) may be superior in some species. Although eastern box turtles’ () hematologic parameters are well studied, the effects of different anticoagulants on hematology in this species are unknown. This study evaluated the effects of LH and EDTA on hematologic values in free-living eastern box turtles ( = 59). Blood samples were collected from eastern box turtles in Illinois and immediately divided between LH and EDTA microtainers, and complete blood counts were performed on each sample. Grossly, plasma from EDTA blood samples was frequently and significantly hemolyzed. Blood mixed with LH had higher packed cell volume (PCV) ( = 0.04), white blood cell count (WBC) determined by Leukopet ( < 0.0001), WBC determined by blood film estimate ( < 0.0001), absolute heterophils ( = 0.007), absolute lymphocytes ( < 0.0001), and lower total solids ( < 0.0001) and absolute monocytes ( = 0.0001) than blood mixed with EDTA. All relative leukocyte counts were significantly different between the anticoagulants ( < 0.0001). EDTA apparently lysed turtle erythrocytes in this study, making it difficult to accurately count white blood cells and artificially lowering PCV. These findings demonstrate that EDTA should not be used in eastern box turtles.