Field grown 2-year-old almond trees (Prunus dulcis cvs. Butte, Carmel, Mission, Ne Plus Ultra, Padre, Peerless, Price, Solano, Sonora, and Thompson) were mechanically inoculated with Xylella fastidiosa in the growing seasons of 2002 and 2003 to study the effect of inoculation date on the movement and colonization of X. fastidiosa and the overwintering persistence of almond leaf scorch disease (ALS) in these cultivars. X. fastidiosa was inoculated into the base of current-season growing shoots in April, May, June, July, August, September, and October. Almond trees inoculated in spring months developed more ALS-symptomatic leaves and more extensive within-plant spread of X. fastidiosa by the end of the current growing season compared with trees inoculated in July, August, September, and October. Trees inoculated in June developed the most severe ALS symptoms during the season in which they were inoculated. Trees inoculated in June and July 2002 had significantly higher disease ratings in 2003 than inoculations made in August and October 2002. Based on disease ratings observed in 2003, 1 year after inoculation, Sonora and Solano were the most ALS susceptible, Mission and Price intermediate, and Carmel, Padre, Ne Plus Ultra, Butte, Peerless, and Thompson were the least susceptible cultivars for allowing X. fastidiosa to overwinter and cause disease the following year. Assessment of all trees in August 2004 indicated that trees inoculated in June and July 2002 had a significantly higher amount of ALS-infected branches than trees inoculated in other months. Butte, Carmel, Padre, and Thompson cultivars had no symptomatic branches, while X. fastidiosa infections persisted or colonized new branches in Sonora, Solano, Peerless, Price, Mission, and Ne Plus Ultra. Based on the 2004 assessment, Sonora was the most susceptible cultivar. Surveys of a diseased orchard in Chico, CA showed large differences in ALS incidence in four almond cultivars. Nonpareil and Peerless had significantly greater incidence of disease than Butte and Carmel over the 2 years surveyed. These data suggest that cultivar susceptibility and the time of X. fastidiosa infection are important factors in determining the persistence of ALS in almond trees.