Racial/ethnic minorities are more vulnerable to mental and physical health problems, but we know little about the psychobiological underpinnings of these disparities. In this study, we examined racial/ethnic differences in cortisol diurnal patterns and affect as initial steps toward elucidating long-term health disparities. A racially/ethnically diverse (39.5% White, 60.5% minority) sample of 370 adolescents (57.3% female) between the ages of 11.9 and 18 years (M = 14.65 years, SD = 1.39) participated in this study. These adolescents provided 16 cortisol samples (4 samples per day across 4 days), allowing the computation of diurnal cortisol slopes, the cortisol awakening response, and diurnal cortisol output (area under the curve), as well as daily diary ratings of high-arousal and low-arousal positive and negative affect. Consistent with prior research, we found that racial/ethnic minorities (particularly African American and Latino youth) exhibited flatter diurnal cortisol slopes compared to White youth, F (1, 344.7) = 5.26, p = .02, effect size g = 0.25. Furthermore, African American and Asian American youth reported lower levels of positive affect (both high arousal and low arousal) compared to White youth. Racial/ethnic differences in affect did not explain differences in cortisol patterns, suggesting a need to refine our models of relations between affect and hypothalamic–pituitary–adrenocortical activity. We conclude by proposing that a deeper understanding of cultural development may help elucidate the complex associations between affect and hypothalamic–pituitary–adrenocortical functioning and how they explain racial/ethnic differences in both affect and stress biology.