Introduction:The neonate exposed to opioids in utero faces a constellation of withdrawal symptoms postpartum commonly called neonatal opioid withdrawal syndrome (NOWS). The incidence of NOWS has increased in recent years due to the opioid epidemic. MicroRNAs (miRNAs) are small non-coding RNA molecules that play a crucial role in gene regulation. Epigenetic variations in microRNAs (miRNAs) and their impact on addiction-related processes is a rapidly evolving area of research.Methods:The Illumina Infinium Methylation EPIC BeadChip was used to analyze DNA methylation levels of miRNA-encoding genes in 96 human placental tissues to identify miRNA gene methylation profiles as-sociated with NOWS: 32 from mothers whose prenatally opioid-exposed infants required pharmacologic management for NOWS, 32 from mothers whose prenatally opioid-exposed infants did not require treat-ment for NOWS, and 32 unexposed controls.Results:The study identified 46 significantly differentially methylated (FDRp-value ≤ 0.05) CpGs associated with 47 unique miRNAs, with a receiver operating characteristic (ROC) area under the curve (AUC) ≥0.75 including 28 hypomethylated and 18 hypermethylated CpGs as potentially associated with NOWS. These dysregulated microRNA methylation patterns may be a contributing factor to NOWS pathogenesis.Conclusion:This is the first study to analyze miRNA methylation profiles in NOWS infants and illustrates the unique role miRNAs might have in diagnosing and treating the disease. Furthermore, these data may provide a step toward feasible precision medicine for NOWS babies as well.