Due to next-generation sequence technologies, sequencing of bacterial genomes is no longer one of the main bottlenecks in bacterial research and the number of new genomes deposited in public databases continues to increase at an accelerating rate. Among these new genomes, several belong to the same species and were generated for pan-genomic studies. A pan-genomic study allows investigation of strain phenotypic differences based on genotypic differences. Along with a need for good assembly quality, it is also fundamental to guarantee good functional genome annotation of the different strains. In order to ensure quality and standards for functional genome annotation among different strains, we developed and made available PANNOTATOR (http://bnet.egr.vcu.edu/iioab/agenote.php), a web-based automated pipeline for the annotation of closely related and well-suited genomes for pan-genome studies, aiming at reducing the manual work to generate reports and corrections of various genome strains. PANNOTATOR achieved 98 and 76% of correctness for gene name and function, respectively, as result of an annotation transfer, with a similarity cut-off of 70%, compared with a gold standard annotation for the same species. These results surpassed the RAST and BASys softwares by 41 and 21% and 66 and 17% for gene name and function annotation, respectively, when there were reliable genome annotations of closely related species. PANNOTATOR provides fast and reliable pan-genome annotation; thereby allowing us to maintain the research focus on the main genotype differences between strains.