Amphibians around the globe must survive physiological stress, whether it originates from chemical pollutants, food shortages, or predation. This physiological stress may cause changes in phenotypes as well as health and performance issues, leading to population declines and ultimately extinction. The Buck Creek Serpentine Barrens (BCSB) in the Nantahala National Forest, Clay County, North Carolina is a unique ecosystem with shallow ultramafic bedrock dominated by pitch pine trees and herbaceous grasses, unlike the surrounding hardwood forests. Seal salamanders (Desmognathus monticola) found at the BCSB often exhibit abnormal yellow patches on their heads, backs, legs, and tails, despite being genetically similar to nearby populations. Despite previous research, the cause of this population’s unique appearance is not understood and was hypothesized to be associated with chronic stress exposure from malnutrition and exposure to harsh serpentine barrens conditions. Analysis of physiological stress requires studying corticosterone (CORT) concentrations, typically using plasma samples. In all but the largest salamander species, however, the collection of plasma samples is usually a terminal procedure. Waterborne hormone analysis is a nonlethal alternative method to plasma sampling that involved submerging sampled individuals into water baths to allow hormones to diffuse across their skin and gills into the water, which is then analyzed for CORT instead of plasma. However, this technique requires validation for each species. In this study, I validated waterborne hormone analysis for seal salamanders using paired water bath and plasma hormone samples. I then used waterborne hormone analysis to investigate whether the unique coloration of the BCSB seal salamander population was associated with high CORT concentrations. I observed no difference in average CORT concentration among the BCSB and neighboring populations, however. Although there were no differences in CORT among populations, I detected significantly higher magnesium concentrations, iron concentrations and water temperatures, as well as decreased calcium concentrations, canopy cover, and macroinvertebrate abundance at the BCSB. These findings document the unusual conditions seal salamanders must overcome to survive at the BCSB. This work provides insight on the effects of serpentine barrens and landscape heterogeneity within the Southern Appalachian Mountains on seal salamander phenotypic diversity and demonstrates the use of a nonlethal method for quantifying CORT in Desmognathus.