Pecan bacterial leaf scorch (PBLS), caused byXylella fastidiosahas been reported in Georgia, Louisiana, Texas, New Mexico, Arizona, and California. Accurate methods are critical for the early detection ofX. fastidiosa, but the validation of current diagnostic tools for pecan has yet to be investigated. Collected petioles, leaflets, and shoots from pecan and otherCaryarelatives in Texas, Indiana, and Georgia were used as tissue samples, and to isolate crude xylem sap and gDNA for side-by-side testing using immunological (ELISA) and molecular-based assays [traditional PCR and real-time quantitative PCR (qPCR)]. Isolated crude sap was found to be the most reliable template for ELISA diagnostics.X. fastidiosa-specific genes were amplified with previously published PCR primer sets; however, they revealed non-specific binding. NewXylella-specific primers were subsequently generated and validated using infected tissue from pecan and relatedCaryaspecies. Two new primer sets (NMU3 and 5) produced expected amplicons specific toX. fastidiosabut did not amplify any non-specific bands of the pecan gDNA. When compared to that of total gDNA as the template in PCR reactions, diluted crude sap was found to be an efficient way to detectX. fastidiosain pecan petioles. A novel TaqMan qPCR assay was also developed for the detection ofX. fastidiosa.The results of the qPCR experiments were equivalent to the traditional PCR amplification when crude sap was used as the template. Comparative PCR analysis confirms that the PCR protocol outlined in this study can be replicated across different laboratories.