Studies on the effects of anticoagulants on blood samples in reptiles are limited. Eastern indigo snakes (Drymarchon couperi) have unique physiological features that are still not well understood. The objective of this study was to evaluate the effects of lithium heparin, tripotassium ethylendiaminetetraacetic acid (EDTA), trisodium citrate, and the use of no anticoagulant on various hematological analytes of the eastern indigo snake. Blood was collected from 10 female eastern indigo snakes and transferred in aliquots into three anticoagulants; it was also processed without anticoagulant. Packed cell volume (PCV), total solids (TS), plasma color, and data from blood film evaluations were compared. Significant differences were found for PCV (P < 0.0001) and TS (P < 0.0001). Both analytes were significantly lower in citrated samples, and heparinized samples had lower TS than EDTA-preserved and non-anticoagulated samples. Although there were no differences in absolute numbers and morphological quality among treatments, heparinized blood samples had a higher heterophil percentage (P = 0.039) compared to non-anticoagulated samples, and both heparinized and EDTA-preserved samples had lower azurophil percentages (P = 0.039) than non-anticoagulated samples. Hemolysis was absent in all samples. Heparinization produced a blue background hue on blood films that was absent in all other methods. This preliminary study demonstrates that heparinized, EDTA-preserved, and non-anticoagulated blood samples are equally superior for hematological evaluation over citrated samples because of its effect on the PCV. This finding has clinical application and it is advised to avoid citrate as an anticoagulant in eastern indigo snakes.