The causal agent of diseases in many economically important plants is attributed to the xylem-limited bacterium Xylella fastidiosa. The detection of this plant pathogen has been hampered due to its difficult isolation and slow growth on plates. Nearly complete nucleotide sequences of the 16S rRNA gene and partial sequences of the gyrB gene were determined for 18 strains of X. fastidiosa isolated from different plant hosts. A phylogenetic analysis, based on gyrB, grouped strains in three clusters; grape-isolated strains formed one cluster, citrus-coffee strains formed another cluster, and a third cluster resulted from all other strains. Primer pairs designed for the 16S rRNA and gyrB genes were extensively searched in databases to verify their in silico specificity. Primer pairs were certified with 30 target and 36 nontarget pure cultures of microorganisms, confirming 100% specificity. A multiplex PCR protocol was developed and its sensitivity tested. Sequencing of PCR products confirmed the validity of the multiplex PCR. Xylella fastidiosa was detected in field-collected plants, disease vector insects, and nonsymptomatic but infected plants. Specific detection of X. fastidiosa may facilitate the understanding of its ecological significance and prevention of spread of the disease.