Background Hair cortisol concentration (HCC) is an increasingly used measure of systemic cortisol concentration. However, determinants of HCC in children and adolescents are unclear because few prospective studies have been conducted to date. Study design We followed 725 children in Project Viva, a pre-birth cohort study of mothers and children, who provided hair samples at mid-childhood (median age: 7.7 years) or early adolescence (median age: 12.9 years). We examined associations of various factors measured from pregnancy to mid-childhood with HCC in mid-childhood and early adolescence, as well as change in HCC between these time points (ΔHCC). Results There were 426 children with HCC measurements in both mid-childhood and early adolescence, 173 children with measures only in mid-childhood, and 126 with measures only in early adolescence. HCC was lower in mid-childhood (median 1.0pg/mg [interquartile range, IQR: 0.5, 2.4]) than early adolescence (2.2pg/mg [1.1, 4.4]). In multivariable-adjusted regression models, female sex (β =-0.41, 95% CI:-0.67,-0.15) and birth weight-for-gestational age z-score (β =-0.19, 95% CI:-0.33,-0.04) were associated with lower mid-childhood HCC, while prenatal smoking was associated with higher mid-childhood HCC (β = 0.53, 95% CI: 0.04, 1.01). In early adolescence, child age (β = 0.34 per year, 95% CI: 0.21, 0.46) female sex (β = 0.33, 95% CI: 0.10, 0.57), and maternal pre-pregnancy body mass