To better understand the processes contributing to symptoms and resistance to Pierce's disease of grape, we examined the movement and multiplication of a green fluorescent protein-marked strain of Xylella fastidiosa in the sterns and petioles of Cabernet Sauvignon, Chenin Blanc, Roucaneuf, and Tampa grape cultivars that differ in their susceptibility to this disease. X. fastidiosa achieved much lower population sizes and colonized fewer xylem vessels in the stem of resistant cultivars compared with more susceptible cultivars. In contrast, X. fastidiosa achieved similarly high population sizes and colonized a similar proportion of the vessels in petioles of susceptible and resistant cultivars, suggesting that, compared with the stem, X. fastidiosa is relatively unrestricted in its movement and growth within the petiole. There was not a direct relationship between the population size of X. fastidiosa in the stem and the proportion of vessels colonized; a much higher population size of the pathogen was observed in susceptible cultivars than expected based on the proportion of vessels colonized. The high population sizes of X. fastidiosa in stems of susceptible genotypes were associated with both a high number of infected vessels and a much higher extent of colonization of those vessels that become infested than in more resistant cultivars. The formation of large cellular aggregates in vessels is not required for X. fastidiosa to move laterally in the stem to adjacent vessels because most vessels harbored only small assemblages, especially in resistant cultivars such as Roucaneuf, in which some intervessel movement was detected. Resistance to Pierce's disease is apparently not due to inhibitory compounds that circulate in the xylem because they might be expected to operate similarly in all tissues.