The bacterium Xylella fastidiosa Wells et al. induces Pierce's disease (PD) of grapevine. This study was initiated to improve sampling protocols to identify X. fastidiosa-infected grapevines (Vitis vinifera L.) in California vineyards. Several potential PD symptoms, including leaf necrosis and chlorosis, internodal distance, petiole length and weight, and extent of cane branching, were not reliable indicators of X. fastidiosa infection. The matchstick symptom (i.e., abscised leaf blades leaving behind a dried, burnt-appearing petiole tip) was the only consistent indicator of infection in X. fastidiosa-positive grapevines. Further study revealed that leaves selected from the most basal nodes of positive canes had the highest probability of X. fastidiosa detection, with enzyme-linked immunosorbent assay. A symptom reliability index (SRI) was created to assess visual PD diagnosis by node location. The SRI values were the highest at basal node locations, but symptoms at any single node were not consistently reliable for PD diagnosis. Our results showed that PD diagnosis based on foliar symptoms was unreliable. However, taking samples from the basal portion of a cane increased the probability of X. fastidiosa detection.