Summary Increased hair cortisol concentrations have been associated with stress exposure in both human and nonhuman primates, and hair cortisol is now gaining attention as a biomarker for stress-related health problems. The present study examined the behavioral and physiological reactions of rhesus monkey (Macaca mulatta) infants reared in three different rearing environments to the major stressor of relocation. Infant monkeys (n=61) were studied from birth through 2 years of age. For the first 8 months of life, infants were either with their mothers and peers (MPR, n=21) or reared in a nursery using either peer-rearing (PR, n=20) or surrogate-peer-rearing (SPR, n=20). At approximately 8 months of age, infants were removed from their rearing group, simultaneously placed into a large social environment consisting of infants from all three rearing conditions, and observed for the next 16 months. Behavior was recorded twice per week from 1 to 24 months, and composite anxiety scores were calculated for each monkey. Monkeys were initially shaved at the nape of the neck on day 14 to remove any prenatal effects on hair cortisol deposition. Hair samples were then collected by re-shaving at 6, 12, 18 and 24 months and analyzed for cortisol content. MPR monkeys were the least affected by the stressor, showing smaller increases in anxious behavior than the other groups and more rapid physiological adaptation as assessed using hair cortisol. PR monkeys showed heightened and prolonged anxious behavior, had the highest cortisol levels prior to relocation, and their cortisol levels did not decline until more than a year later. SPR monkeys exhibited more rapid behavioral adaptation than PR monkeys, showing heightened but not prolonged anxious behavior. However, the SPR group showed a marked increase in cortisol in response to the relocation, and like the PR group, their physiological adaptation was slower than that of the MPR group as indicated by elevated cortisol levels at 18 months. By 24 months of age (16 months after relocation), all rearing groups were indistinguishable from one another physiologically and behaviorally. Spearman rank correlation revealed that hair cortisol taken at month 6 was not correlated with composite anxiety scores from months 6 to 8 (just before the relocation), but was positively correlated with composite anxiety scores between months 8 and 12 (immediately after relocation) for PR infants only (rs=0.75, p<0.001). Month 6-hair cortisol tended to positively correlate with composite anxiety scores for the following 6 months (months 12–18) for PR monkeys only (rs=0.47, p=0.037), which exhibited more anxious behavior than MPR and SPR infants during this period (ANOVA: F(2,60)=14.761, p<0.001) This is the first study to show that elevated hair cortisol early in life is a biomarker for the later development of anxious behavior in response to a major life stressor, particularly for infant monkeys exposed to early life adversity in the form of peer-rearing.