Recent advances in the development of high-throughput tools have significantly revolutionized our understanding of molecular mechanisms underlying normal and dysfunctional biological processes. Here we present a novel computational tool, transcription factor search and analysis tool (TrFAST), which was developed for the in silico analysis of transcription factor binding sites (TFBSs) of signaling pathway-specific TFs. TrFAST facilitates searching as well as comparative analysis of regulatory motifs through an exact pattern matching algorithm followed by the graphical representation of matched binding sites in multiple sequences up to 50kb in length. TrFAST is proficient in reducing the number of comparisons by the exact pattern matching strategy. In contrast to the pre-existing tools that find TFBS in a single sequence, TrFAST seeks out the desired pattern in multiple sequences simultaneously. It counts the GC content within the given multiple sequence data set and assembles the combinational details of consensus sequence(s) located at these regions, thereby generating a visual display based on the abundance of unique pattern. Comparative regulatory region analysis of multiple orthologous sequences simultaneously enhances the features of TrFAST and provides a significant insight into study of conservation of non-coding cis-regulatory elements. TrFAST is freely available at http://www.fi-pk.com/trfast.html.