In the context of DNA Barcoding, sequences of standard marker genes for thousands and potentially millions of individuals and species are becoming available, requiring ever more efficient bioinformatic environments and software algorithms for analysis. We here present ExcaliBAR (Extraction, Calculation, Barcoding), a user-friendly software utility to facilitate one important initial step in DNA barcoding analyses, namely the determination of the barcoding gap between pairwise genetic distances among and within species, based on original distance matrices computed by MEGA software. In addition, the software is able to rename sequences downloaded via the standard user interfaces of public databases such as GenBank, without the need of developing and applying specific scripts for this purpose.