The xylem-limited bacterium Xylella fastidiosa is the causal agent of Pierce's disease (PD) in grapevines, for which breeding resistant cultivars will be a long-term management strategy that involves the identification and characterization of resistant germplasm. A genetically diverse group of grape species and selections was mechanically inoculated with X. fastidiosa, grown in a greenhouse for 113 days after inoculation, and evaluated for the levels of bacterial concentrations in stem and leaf tissues by quantitative enzyme-linked immunosorbent assay (ELISA). Concentrations of X. fastidiosa were affected by genotype, plant tissue, position on the plant relative to the point of inoculation, and interactions among these factors. Based on estimated concentrations of X. fastidiosa in stem samples at 113 d postinoculation, 9621-67, Muscadinia rotundifolia, Vitis arizonica/candicans, V arizonica/girdiana, V candicans, V girdiana, V nesbittiana, and V shuttleworthii were resistant to PD. In contrast, V vinifera, V aestivalis, 9621-94, and V champinii had very high X. fastidiosa concentrations in stem tissues. Sequential sampling of leaf blades at 34, 77, and 113 d postinoculation revealed different temporal patterns in X. fastidiosa concentrations among the grape genotypes. Estimates of X.fastidiosa concentrations decreased after the first sampling in M. rotundifolia, 9621-67, V girdiana, and V arizonica/candicans but increased in all other genotypes. The characterization of X. fastidiosa concentrations in a broad range of grape genotypes allows for the selection of promising genetic backgrounds capable of greatly limiting the population size and development of X. fastidiosa in stems, a critical trait in the breeding of PD-resistant grapevines.