Fluorescence microscopy, enzyme-linked immunosorbent assay, and direct culturing were used to determine natural infection of various weeds and wild plants with Xylella fastidiosa, a xylem-limited bacterium. Strains of X. fastidiosa that could produce Pierce’s disease symptoms in inoculated grapevine were cultured from American elder (Sambucus canadensis), Virginia creeper (Parthenocissus quinquefolia), peppervine (Ampelopsis arborea), American beautyberry (Callicarpa americana), and blackberry (Rubus sp.). These were cultured on PD3 medium, originally developed for the PD strain bacterium. Using PW medium, strains of X. fastidiosa that were not pathogenic to grapevine and would not grow on PD3 medium were cultured from eastern baccharis (Baccharis halimifolia), sumac (Rhus sp.), goldenrod (Solidago fistulosa), southern red oak (Quercus falcata), laurel oak (Q. laurifolia), water oak (Q. nigra), peach (Prunus persica), and sycamore (Platanus occidentalis). A large number of natural hosts of X. fastidiosa occur in Florida, and there is a need to study pathological and taxonomic relationships among the strains of the bacterium.