Hellbenders are large aquatic salamanders. The Ozark subspecies is in decline through its range. This is the first comprehensive report on hematologic and serum chemistry for both Ozark and eastern hellbenders. Blood samples were analyzed for 25 parameters in 33 Ozark hellbenders from the North Fork of White River and the Eleven Point River in Missouri and 45 eastern hellbenders from the Davidson River-Looking Glass Creek in North Carolina and the Cooper Creek in Georgia. Each river was considered a population. In general, the majority of the blood parameters analyzed were similar between populations and subspecies for same-sex individuals, although a few significant differences were identified. The baseline data we acquired are important for future monitoring of hellbender populations, particularly as Ozark hellbender populations continue to age.