RSAT (Regulatory Sequence Analysis Tools) is a suite of modular tools for the detection and the analysis of cis-regulatory elements in genome sequences. Its main applications are (i) motif discovery, including from genome-wide datasets like ChIP-seq/ATAC-seq, (ii) motif scanning, (iii) motif analysis (quality assessment, comparisons and clustering), (iv) analysis of regulatory variations, (v) comparative genomics. Six public servers jointly support 10 000 genomes from all kingdoms. Six novel or refactored programs have been added since the 2015 NAR Web Software Issue, including updated programs to analyse regulatory variants (retrieve-variation-seq, variation-scan, convert-variations), along with tools to extract sequences from a list of coordinates (retrieve-seq-bed), to select motifs from motif collections (retrieve-matrix), and to extract orthologs based on Ensembl Compara (get-orthologs-compara). Three use cases illustrate the integration of new and refactored tools to the suite. This Anniversary update gives a 20-year perspective on the software suite. RSAT is well-documented and available through Web sites, SOAP/WSDL (Simple Object Access Protocol/Web Services Description Language) web services, virtual machines and stand-alone programs at http://www.rsat.eu/.