ABSTRACTTranscription factors (TF) regulate gene expression by binding to specific sequences known as motifs. A bottleneck in our knowledge of gene regulation is the lack of functional characterization of TF motifs, which is mainly due to the large number of predicted TF motifs, and tissue specificity of TF binding. We built a framework to identify tissue-specific functional motifs (funMotifs) across the genome based on thousands of annotation tracks obtained from large-scale genomics projects including ENCODE, RoadMap Epigenomics and FANTOM. The annotations were weighted using a logistic regression model trained on regulatory elements obtained from massively parallel reporter assays. Overall, genome-wide predicted motifs of 519 TFs were characterized across fifteen tissue types. funMotifs summarizes the weighted annotations into a functional activity score for each of the predicted motifs. funMotifs enabled us to measure tissue specificity of different TFs and to identify candidate functional variants in TF motifs from the 1000 genomes project, the GTEx project, the GWAS catalogue, and in 2,515 cancer samples from the Pan-cancer analysis of whole genome sequences (PCAWG) cohort. To enable researchers annotate genomic variants or regions of interest, we have implemented a command-line pipeline and a web-based interface that can publicly be accessed on: http://bioinf.icm.uu.se/funmotifs.