Excessive prenatal opioid exposure may lead to the development of Neonatal Opioid Withdrawal Syndrome (NOWS). RNA-seq was done on 64 formalin-fixed paraffin-embedded placental tissue samples from 32 mothers with opioid use disorder, with newborns with NOWS that required treatment, and 32 prenatally unexposed controls. We identified 93 differentially expressed genes in the placentas of infants with NOWS compared to unexposed controls. There were 4 up- and 89 downregulated genes. Among these, 7 genes CYP1A1, APOB, RPH3A, NRXN1, LINC01206 , AL157396.1 , UNC80 achieved an FDR p -value of <0.01. The remaining 87 genes were significant with FDR p-value <0.05. The 4 upregulated, CYP1A1 , FP671120.3, RAD1, RN7SL856P, and the 10 most significantly downregulated genes were RNA5SP364, GRIN2A, UNC5D, DMBT1P1, MIR3976HG, LINC02199, LINC02822, PANTR1, AC012178.1, CTNNA2. Ingenuity Pathway Analysis identified the 7 most likely to play an important role in the etiology of NOWS. Our study expands insights into the genetic mechanisms of NOWS development. • We performed a transcriptome analysis to determine the pathophysiology and development of Neonatal Opioid Withdrawal Syndrome • This study identified 93 differentially expressed genes and 7 dysregulated pathways that could influence NOWS progression • Netrin Signaling, CREB Signaling in Neurons, Calcium, Synaptogenesis, and Opioid Signaling are top-ranking IPA pathways. • This study provides insight into the molecular mechanisms underlying NOWS and provides a basis for further critical research. [ABSTRACT FROM AUTHOR]