The ultimate goal of metagenome research projects is to understand the ecological roles and physiological functions of the microbial communities in a given natural environment. The 454 pyrosequencing platform produces the longest reads among the most widely used next generation sequencing platforms. Since the relatively longer reads of the 454 platform provide more information for identification of microbial sequences, this platform is dedicated to microbial community and population studies. In order to accurately perform the downstream analysis of the 454 multiplex datasets, it is necessary to remove artificially designed sequences located at either ends of individual reads and to correct low-quality sequences. We have developed a program called PyroTrimmer that removes the barcodes, linkers, and primers, trims sequence regions with low quality scores, and filters out low-quality sequence reads. Although these functions have previously been implemented in other programs as well, PyroTrimmer has novelty in terms of the following features: i) more sensitive primer detection using Levenstein distance and global pairwise alignment, ii) the first stand-alone software with a graphic user interface, and iii) various options for trimming and filtering out the low-quality sequence reads. PyroTrimmer, written in JAVA, is compatible with multiple operating systems and can be downloaded free at http://pyrotrimmer.kobic.re.kr.