We describe a fast, automated process to determine distances between mtDNA sequences allowing their subsequent clustering and haplogroup assignment that may increase the speed of data analysis and avoid human errors. In order to avoid complexities/ambiguities resulting from recurrence and insertion/deletion phenomena and thus improving evolutionary signal-to-noise ratio, protein coding sequences were compared using a vectorial representation method, and the corresponding genetic distance matrix was used for the construction of a neighbor-joining/UPGMA tree or an MDS graphic, which generally agrees with the consensus mtDNA phylogeny. mtDNAoffice software, detailed instructions and example files are freely available on the web at http://www.portugene.com/SupMat/setupmtDNAoffice.rar.