ObjectiveTo compare brain MRI biomarkers and neurodevelopmental test scores in infants born preterm with and without prenatal opioid exposure (POE). Study designWe examined 395 preterm infants (≤ 32 weeks gestational age[GA]) who had term-equivalent brain MRIs, composite scores from the Bayley Scales of Infant and Toddler Development-III(BSID-III) at 2 years corrected age, and POE data. MRI parameters included total/regional brain volumes and severe punctate white matter lesions (PWMLs). We conducted bivariable analysis and multivariable logistic regression analyses. ResultsThe mean(SD) gestational age was 29.3(2.5) weeks; 35(8.9%) had POE and 20(5.1%) had severe PWML. Compared with unexposed infants, those with POE exhibited higher rates of severe PWML (17.1% vs. 3.9%, respectively; p=0.002); findings remained significant with OR 4.16 (95% CI:1.26, 13.68) after adjusting for confounders. On mediation analysis, the significant relationship between POE and severe PWML was not indirectly mediated through preterm birth/GA (OR:0.93; 95% CI:0.78,1.10), thus suggesting the association was largely driven by a direct adverse effect of POE on white matter. In multivariable analyses, POE was associated with a significantly lower score by -6.2 (95%CI: -11.8,-0.6) points on BSID-III Motor subscale compared with unexposed infants. ConclusionsPOE was associated with severe PWML; this outcome may be a direct effect of POE rather than being mediated by premature birth. POE was also associated with worse motor development. Continued follow-up to understand the long-term effects of POE is warranted.