Evaluating leukocytes provides insight in the overall health of animals by characterizing pathologies related to infection, inflammation, and stress. In reptile species, many leukocyte quantification methods are used to evaluate white blood cell (WBC) parameters; however, agreement and analytical variability (repeatability) between the various methods have yet to be reported and critically assessed in many species, including the prairie rattlesnake (Crotalus viridis). The objectives of this study were to 1) test agreement between hematology parameters determined by phloxine-based stain (Eosinophilic leukopet, EO), Natt–Herrick's (NH), and estimate (EST) methods using fresh whole blood and lithium-heparinized blood; 2) determine the agreement between estimates from a veterinary student and from a board-certified clinical pathologist; and 3) determine the analytical variability of EO, NH, and EST methods. Venipuncture was performed in 27 adult snakes, and agreement was then assessed using Bland–Altman plots and Passing–Bablock regression between all methods. There was a variable degree of proportional and constant error between many of the quantification methods, but descriptive statistics for all methods fell within biologically relevant values. There was good agreement in the EST method between the clinical pathologist and the veterinary student, but the student had a significantly higher mean WBC count in fresh whole blood. The lowest analytical variability was observed with the NH method (15.4%), whereas the highest was observed with the EST method (58.6% for the clinical pathologist, 59.4% for the veterinary student). These results identify the intrinsic variability in reptile hematological methods currently used and emphasize the need to develop a singular diagnostic standard for leukocyte quantification in reptile species.