American alligators, Alligator mississippiensis, are one of 23 crocodilian species in the world, and they are common in the southeastern United States. Biochemical enzyme blood reference values have been established for the American alligator and several other crocodilians, but the tissue of origin of these enzymes has not been established. This study evaluated tissue specificity of six different enzymes in the American alligator, including alkaline phosphatase (ALP), alanine aminotransferase (ALT), aspartate aminotransferase (AST), lactate dehydrogenase (LDH), creatine kinase (CK), and gamma-glutamyl transferase (GGT). Six different tissues (liver, cardiac muscle, lung, kidney, skeletal muscle, and mid–small intestine) were collected from six subadult American alligators for enzyme analysis. The results showed that the CK and LDH enzymes were primarily derived from skeletal and cardiac muscle; ALT was derived primarily from kidney and skeletal muscle tissue; and AST was derived primarily from liver and skeletal muscle tissue. GGT and ALP appear to be of little diagnostic value regarding the tissues sampled because there was no statistical difference between the tissue levels. The results of this study should be of use to clinicians when interpreting biochemistry enzyme levels from blood samples.