A three-stranded nucleic acid structure is universally formed in the process of transcription, as the nascent RNA molecule will hybridize with the template DNA strand forming a DNA:RNA hybrid and leaving the non-templated DNA single stranded. Such structures are termed as R-loops and have been found in various organisms with essential physiological functions. However, we still have limited understanding of formation and preferences, as well as the biological functions of R-loops in plant genomes. By using the newly developed ssDRIP-seq, an efficient, unbiased, high through-put and strands-specific R-loop profiling method, we have been able to identify thousands of R-loop loci in plant genomes. Comprehensive analysis showed that R-loops are strongly correlated with many chromatin modifications. Our data suggests R-loop is the common feature of Arabidopsis genome organization. I will present our most recent results about genome-wide R-loops characterization, as well as their biological functions in plant development.