Xylella fastidiosa causes many economically important plant diseases such as Pierce's disease of grapevine, citrus variegated chlorosis disease, and olive quick decline syndrome. Another species in the same genus, Xylella taiwanensis, causes pear leaf scorch. Here, to enable an initial screening of plants suspected of being infected with Xylella spp. by conventional polymerase chain reaction (cPCR), new primer pairs—X67S1/XL2r and XrDf1/XLr4—were designed to target the 16S ribosomal DNA (rDNA) of not only X. fastidosa but also X. taiwanensis. In cPCR to detect both species, X67S1/XL2r showed features superior to those of other primer pairs, such as fewer false negatives and false positives, whereas XrDf1/XLr4 seemed to be unsuitable because of abundant non-specific amplification. However, when XrDf1/XLr4 was combined with a probe in a TaqMan quantitative real-time PCR (qPCR), the assay detected no false positives and was more useful in the universal detection of Xylella spp. than TaqMan qPCR assays reported previously.