Abstract Summary: With the rapid development of DNA sequencing technology, increasing bacteria genome data enable the biologists to dig the evolutionary and genetic information of prokaryotic species from pan-genome sight. Therefore, the high-efficiency pipelines for pan-genome analysis are mostly needed. We have developed a new pan-genome analysis pipeline (PGAP), which can perform five analytic functions with only one command, including cluster analysis of functional genes, pan-genome profile analysis, genetic variation analysis of functional genes, species evolution analysis and function enrichment analysis of gene clusters. PGAP's performance has been evaluated on 11 Streptococcus pyogenes strains. Availability:PGAP is developed with Perl script on the Linux Platform and the package is freely available from http://pgap.sf.net. Contact: junyu@big.ac.cn; xiaojingfa@big.ac.cn Supplementary information: Supplementary data are available at Bioinformatics online.