Cloned DNA's from the region between the 16S rRNA and 23S rRNA genes of Clavibacter xyli subsp. xyli, the causal bacterium of ratoon stunting disease of sugarcane, and from the albicidin gene complex of Xanthomonas albilinians, the causal bacterium of leaf scald disease of sugarcane, were sequenced, and the sequences used to design PCR primers for the detection of the two pathogens. Different combinations of paired primers were examined for detection of both pathogens in single multiplex reactions. When purified total DNA of each pathogen were tested, a minimum of 25 pg or less DNA was detected with most primer pairs; however, detection limit of multiplex PCR were adversely affected when DNA of the two pathogens were present in disproportionate amounts. Some primers or combinations of primers produced unexpected products in multiplex PCR. The relative sensitivity of detection for primer pairs varied for detection of the pathogens in sugarcane extracts, apparently because plant DNA or PCR inhibitors in the extracts had a differential effect on PCR with different primers. Some combinations of primers also produced PCR products when bacterial combinations isolated from sugarcane were tested. PCR detection of the pathogens, in both single and multiplex reactions, was improved by careful selection of PCR primers and reaction parameters.