BACKGROUND: Annotated phylogenetic trees that display the evolution of transcription factor binding in regulatory regions are useful for e.g. 1) narrowing down true positive predicted binding sites, providing predictions for binding sites that can be tested experimentally, and 2) giving insight into the evolution of gene regulation and regulatory networks. RESULTS: We describe ReXSpecies, a web-server that processes the sequence information of a regulatory region for multiple species and associated (predicted) transcription factor binding sites into two figures: a) An annotated alignment of sequence and binding sites, consolidated and filtered for ease of use, and b) an annotated tree labeled by the gain and loss of binding sites, where the tree can be calculated from the data or taken from a trusted taxonomy, and the labels are calculated based on standard or Dollo parsimony. For genes involved in mammalian pluripotency, ReXSpecies trees highlight useful patterns of transcription factor binding site gain and loss, e.g. for the Oct and Sox group of factors in the 3' untranslated region of the cystic fibrosis transmembrane conductance regulator gene, which closely match experimental data. CONCLUSION: ReXSpecies post-processes the information provided by transcription factor binding site prediction tools, in order to compare data from many species. The tool eases visualization and successive interpretation of transcription factor binding data in an evolutionary context. The ReXSpecies URL can be found in the Availability and requirements section.