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Neonatal abstinence syndrome (NAS) after in‐utero opioid exposure remains a poorly understood condition with multiple factors contributing to severity. Exposure to maternal stress may be one contributing factor. Hair cortisol measurement represents a novel technique for assessing prenatal stress. In this pilot study, the association between maternal hair cortisol levels and NAS severity was examined in 70 postpartum women with opioid use disorder within 72 hr of delivery. Infants were monitored for NAS and treated according to institutional protocol. Forty‐four (63%) of the infants were pharmacologically treated for NAS, with a mean length of hospital stay (LOS) for all infants of 14.2 (SD9.0) days. The mean cortisol level in the mothers was 131.8 pg/mg (SD124.7). In bivariate analysis, higher maternal hair cortisol levels were associated with shorter infant LOS (R = −.26,p = .03) and fewer infant opioid treatment days (R = −.28,p = .02). Results were no longer statistically significant in regression models after adjusting for maternal opioid and smoking. In conclusion, we demonstrated the feasibility of hair cortisol assaying within the first few days after delivery in mothers with opioid use disorder as a novel marker for NAS. The findings suggest that maternal stress may impact the severity of infant opioid withdrawal.