Summary: Phylogenetic profiles form the basis for tracing proteins and their functions across species and through time. Novel genome sequences nowadays often represent species from the remotest corner of the tree of life. Thus, phylogenetic profiling becomes increasingly important for functionally annotating this data and to integrate it into a comprehensive view on organismal evolution. To strengthen the link between the sharing of a gene across species and of the corresponding function, it is meanwhile common to complement phylogenetic profiles with additional information, such as domain architecture similarities between orthologs, or pairwise similarities of other protein features. However, there are few visualization tools that facilitate an intuitive integration of these various information layers. Here, we present PhyloProfile, an R-based tool to visualize, explore and analyze multi-layered phylogenetic profiles. Availability and implementation: PhyloProfile is available as open source code under the MIT license at https://github.com/BIONF/phyloprofile. An online version for testing PhyloProfile and for small to medium-scale analyses is available at http://applbio.biologie.uni-frankfurt.de/phyloprofile.