Xylella fastidiosa is a xylem-limited, nutritionally fastidious bacterium that causes several plant diseases including Pierce's disease (PD) in grape and leaf scorch in almond (ALS) and oleander (OLS). OLS strains belong to X. fastidiosa subsp. sandyi, PD strains belong to X. fastidiosa subsp. fastidiosa, and strains from almond designated as ALS strains are of two general types belonging either to X. fastidiosa subsp. multiplex or X. fastidiosa subsp. fastidiosa. The ALS strains assigned to X. fastidiosa subsp. multiplex belong to two different genotypes (ALSI and ALSII) below the subspecies level. The OLS strains do not infect grape or almond. PD strains produce diseases in grape, alfalfa, almond, and some weeds, but they do not infect oleander, oak, peach, or citrus. ALS strains that belong to X. fastidiosa subsp. multiplex do not produce disease on grape. In this study, a relatively simple polymerase chain reaction (PCR) based method was developed to distinguish among PD, OLS, and ALS strains. PCR performed with primers XF1968-L and XF1968-R amplified a 638-bp fragment from OLS strains but not from PD strains or ALS strains that belong to X. fastidiosa subsp. fastidiosa. PCR with primers XF2542-L and XF2542-R amplified a 412-bp fragment from PD strains, but not from OLS strains. PCR with primers ALM1 and ALM2 produced a fragment of 521 bp from strains isolated from almond that belong to X. fastidiosa subsp. multiplex. The combination of the three primer sets allowed the distinction of the two ALS genotypes of X. fastidiosa subsp. multiplex. These results are in agreement with those obtained from analysis of sequences of 16S-23S rDNA intergenic spacer regions sequence analysis and with previous results based on randomly amplified polymorphic DNA analysis.