Double-stranded DNA is able to form triple-helical structures by accommodating a third nucleotide strand in its major groove. This sequence-specific process offers a potent mechanism for targeting genomic loci of interest that is of great value for biotechnological and gene-therapeutic applications. It is likely that nature has leveraged this addressing system for gene regulation, because computational studies have uncovered an abundance of putative triplex target sites in various genomes, with enrichment particularly in gene promoters. However, to draw a more complete picture of the in vivo role of triplexes, not only the putative targets but also the sequences acting as the third strand and their capability to pair with the predicted target sites need to be studied. Here we present Triplexator, the first computational framework that integrates all aspects of triplex formation, and showcase its potential by discussing research examples for which the different aspects of triplex formation are important. We find that chromatin-associated RNAs have a significantly higher fraction of sequence features able to form triplexes than expected at random, suggesting their involvement in gene regulation. We furthermore identify hundreds of human genes that contain sequence features in their promoter predicted to be able to form a triplex with a target within the same promoter, suggesting the involvement of triplexes in feedback-based gene regulation. With focus on biotechnological applications, we screen mammalian genomes for high-affinity triplex target sites that can be used to target genomic loci specifically and find that triplex formation offers a resolution of ∼1300 nt.