Studies are increasingly using cholinergic parameters as biomarkers of early neurotoxicity, but few have characterized this system in ecologically relevant model organisms. In the present study, key neurochemicals in the cholinergic pathway were measured and analyzed from discrete parts of brain and blood from captive mink (Mustela vison). Similar to other mammals, the regional distribution of cholinergic parameters in the brain could be ranked from highest to lowest as: basal ganglia > occipital cortex > brain stem > cerebellum (F (3,192) = 172.1, p < 0.001). Higher variation in cholinergic parameters was found in the cerebellum (coefficient of variation = 34.9%), and the least variation was measured in the brain stem (19.7%). Variation was also assessed by calculating the difference between the lowest and highest measures among individual animals: choline acetyltransferase (1.6x fold difference), cholinesterase (2.0x), muscarinic receptor levels (2.4x), acetylcholine (3.7x), nicotinic receptor levels (3.9x), and choline transporter (5.0x). In blood samples, activity and inter-individual variation of cholinesterase was highest in whole blood and lowest in plasma and serum. By using captive mink of a common genetic source, age, gender, and rearing conditions, these data help establish normal levels, ranges, and variations of cholinergic biomarkers among brain regions, blood components, and individual animals. Such information may better enable the utility of cholinergic biomarkers in environmental assessments.