OBJECTIVE To analyze adverse childhood experiences (ACEs) among mothers of newborns referred to a hospital's child protection team (CPT) for suspected substance exposure. Researchers hypothesized that a higher prevalence of these mothers have ≥ 4 ACEs than female counterparts in the general population. The study team also explored whether associations existed between type of maternal ACEs and substance use in pregnancy. METHODS Retrospective review of infant referrals to the CPT in the 3 years after adding an ACEs questionnaire to the consultation process. Bivariate analyses and multivariate logistic regression models examined associations between prenatal substance use and maternal ACEs prevalence, controlling for demographics. RESULTS Data from 222 infants (four sets of twins) and 218 mothers were analyzed. Half (50.0%) the infants had withdrawal symptoms. Most (67.0%) women had positive toxicology screens, while 85.0% reported prenatal substance use. Half (50.9%) the mothers reported ≥ 4 ACEs and these individuals had significantly higher odds of cannabinoid use [adjusted odds ratio (aOR), 3.7; 95%CI 2.0, 6.9, p < 0.001) than those with < 4 ACEs. A significant association was found between substance use and ACEs in the household challenges category (p = 0.03), especially parental separation/divorce (p < 0.001). CONCLUSIONS As hypothesized, a higher prevalence of mothers referred to the CPT had ≥ 4 ACEs than women in the general population (50.9% vs. 15.2%), and a large proportion had used substances while pregnant. Routine prenatal ACEs screening and universal, nonpunitive toxicology testing of infants and mothers at birth may provide opportunities for intervention while reducing the transgenerational impact of ACEs.