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Background: Multiplex PCR, defined as the simultaneous amplification of multiple regions of a DNA template using more than one primer set (comprising a forward primer and a reverse primer) in one tube, has been widely used in clinical and environmental microbiology studies. However, primer design for multiplex PCR is still a challenging problem and several factors need to be considered. These problems include mis-priming due to nonspecific binding to non-target DNA templates, primer dimerization, and the inability to separate and purify DNA amplicons of similar electrophoretic mobility. Results: A new program named MPprimer was developed to help users design primer sets for multiplex PCR with high reliability. It employs the widely used primer design program Primer3 and the primer specificity evaluation program MFEprimer to design and evaluate the candidate primers based on genomic or transcript DNA database, followed by careful examination to avoid primer dimerization.