Background: The number of available genome sequences is increasing, and easy-to-use software that enables efficient comparative analysis is needed. Results: We developed GenomeMatcher, a stand-alone software package for Mac OS X. GenomeMatcher executes BLAST and MUMmer, and the detected similarities are displayed in twodimensional and parallel views with similarity values indicated by color. Selection and recomputation of any subregions is easily performed and allows flexible and in-depth analysis. Furthermore, symbols for annotation data are displayed along the views, and the user can relate the genomic differences with annotation data. While bl2seq allows sub-Giga base comparison, three alignment programs, bl2seq, MAFFT and ClustalW, together with a dotmatch program allow comparative analysis of single-nucleotide level resolution. GenomeMatcher images can be saved as PDF and TIFF files for presentation. As examples of graphical ability of GenomeMatcher to show similarity in colors, ...