RLSeq is a toolkit for analyzing and evaluating R-loop mapping datasets. RLSeq serves two primary purposes: (1) to facilitate the evaluation of dataset quality, and (2) to enable R-loop analysis in the context of publicly-available data sets from RLBase. The package is intended to provide a simple pipeline, called with the RLSeq() function, which performs all main analyses. Individual functions are also accessible and provide custom analysis capabilities. Finally an HTML report is generated with report().