The Coronavirus Disease 2019 (COVID-19) pandemic has spread across the world and resulted in over 5 million deaths to date, as well as countless lockdowns, disruptions to daily life, and extended period of social distancing and isolation. The impacts on youth in particular are astounding, with shifts in learning platforms, limited social outlets, and prolonged uncertainty about the future. Surveys have shown that mental health among youth has severely suffered during the pandemic. However, limited research to date has reported on physiological indices of stress surrounding the pandemic, such as cortisol. Cortisol is a stress hormone that typically increases during stressful situations and can have deleterious effects on mental and physical health when chronically heightened. The present study leveraged hair cortisol concentration measurements, which allowed the retrospectiveinvestigation of circulating cortisol prior to- versus after pandemic-related local lockdowns during the first wave of the pandemic. A final sample of 44 youth ages 10- to 18-years-old provided hair samples and reported on their perceived affective well-being and level of concern regarding pandemic-related stressors between May and June of 2020. We found significant levels of concern and decreases in affective well-being following local lockdowns. Moreover, we saw that cortisol robustly increased following local lockdowns, and those increases were predictive of changes in affect. These findings provide critical insights into the underlying neuroendocrinology of stress during the pandemic and support the need for resources to support youths’ mental health and well-being during this globally significant event.