Xylella fastidiosa is the xylem-limited bacterium that causes Pierce's disease of grapevine and oleander leaf scorch. Detection of this pathogen prior to symptom development is critical for improved management of the disease. Enzyme-linked immunosorbent assay (ELISA) and polymerase chain reaction (PCR) currently are used for routine detection of the pathogen; however, both detection methods are limited by low titer or patchy distribution of the bacterium within a host plant. In the study reported here, we directly compared X. fastidiosa detection in whole-tissue samples with xylem fluid samples from grapevine and oleander. Collection of xylem fluid samples improved sensitivity of pathogen detection by ELISA (41.0%) compared with whole-tissue samples (20.5%) in asymptomatic grapevine. Additionally, pathogen detection in asymptomatic grapevine by PCR also was improved when xylem samples were tested (66.7%) compared with whole-tissue samples (23.1%). There were no differences in frequency of detection of X. fastidiosa in symptomatic grapevines by ELISA or PCR dependent upon sample collection method. Assays of xylem fluid samples did not improve detection of X. fastidiosa in symptomatic or asymptomatic oleander compared with assays of whole tissue. Finally, in a direct comparison of ELISA and PCR, we found no significant differences in frequencies of positive grapevine or oleander samples detected.