Xylella fastidiosa (Xf), a xylem-limited bacterium, is the causal agent of many plant diseases, including Pierce's disease (PD) of grapevine. Reliable detection of PD depends upon accurate and repeatable methods to isolate and identify Xf in grapevines. A pressure chamber was used to extrude xylem fluids from grapevine shoots. Xf was isolated from xylem exudates of infected shoots, while Xf was not found in xylem exudate from healthy shoots on the same plant. Polymerase chain reaction detected Xf in bacterial suspensions from xylem exudates using either immunocapture or centrifugation to avoid or eliminate polymerase inhibitors. Pressure chamber extraction followed by centrifugation of xylem exudate provides a simple, rapid, and efficient protocol for isolation and detection of Xf. This procedure would greatly aid epidemiological studies of PD that involve large numbers of plant samples.