Conservation biology integrates multiple disciplines to expand the ability to identify threats to populations and develop mitigation for these threats. Road ecology is a branch of conservation biology that examines interactions between wildlife and roadways. Although the direct threats of road mortality and habitat fragmentation posed by roads have received much attention, a clear understanding of the indirect physiological effects of roads on wildlife is lacking. Chronic physiological stress can lower immune function, affect reproductive rates and reduce life expectancy; thus, it has the potential to induce long-lasting effects on populations. Reptiles are globally in decline, and roads are known to have negative effects on reptile populations; however, it is unknown whether individual responses to roads and traffic result in chronic stress that creates an additional threat to population viability. We successfully extracted reliable measures of corticosterone (CORT), a known, commonly used biomarker for physiological stress, from claw trimmings from painted turtles (Chrysemys picta) captured at three study sites (road-impacted site, control site and validation site). Corticosterone levels in claws were evaluated as a measure of chronic stress in turtles because CORT is deposited during growth of the claw and could provide an opportunity to examine past long-term stress levels. While male turtles had higher CORT levels on average than females, there was no difference in the level of CORT between the road-impacted and control site, nor was there a relationship between CORT and turtle body condition. In validating a novel approach for non-invasive measurement of long-term CORT levels in a keratinized tissue in wild reptiles, our study provides a new avenue for research in the field of stress physiology.