R-loops, RNA:DNA hybrid structures formed during transcription, have emerged as critical regulators of gene expression and genome stability. Initially considered byproducts of transcription, R-loops are now recognized as key players in various cellular processes, including transcription regulation, chromatin remodeling, and DNA repair. However, abnormal accumulation of R-loops can lead to excessive DNA damage and genomic instability, contributing to the pathogenesis of various diseases. This chapter provides a comprehensive overview of recent advances in R-loop biology, emphasizing their formation, genomic distribution, and intricate interactions with epigenetic modifications. We discuss how R-loops influence chromatin architecture and gene expression and the mechanisms through which they contribute to genomic instability. Furthermore, we examine the involvement of R-loops in the pathogenesis of neurological diseases, including repeat expansion disorders and autism spectrum disorder (ASD). Finally, we discuss potential therapeutic strategies targeting pathological R-loops, emphasizing the need for precise compounds to selectively mitigate R-loop accumulation without causing off-target effects. This chapter aims to provide an updated perspective on R-loop biology and its significance in neurological disorder research.