R-loops are dynamic three-stranded nucleic acid structures that form naturally during transcription. These structures typically arise when the newly synthesized RNA hybridizes with the DNA template strand, displacing the non-template DNA strand. R-loops are not only found at protein-coding genes but also in regions producing non-coding RNAs, such as telomeres, centromeres, ribosomal DNA genes, and transfer RNA genes. While R-loops are regulated by both the process of transcription and chromatin structures, they also play a critical role in modulating transcription and influencing the chromatin landscape. Moreover, the interactions between R-loops, transcription, and chromatin are essential for maintaining genome stability and are often disrupted in various human diseases. In this review, we will explore recent insights into the intricate relationship between R-loops and transcription, as well as their crosstalk with chromatin.