Xylella fastidiosa is a pathogen that causes leaf scorch and related diseases in over 100 plant species, including Pierce's disease in grapevines (PD), phony peach disease (PP), plum leaf scald (PLS), and leaf scorch in almond (ALS), oak (OAK), and oleander (OLS). We used a high-resolution DNA sequence approach to investigate the evolutionary relationships, geographic variation, and divergence times among the X. fastidiosa isolates causing these diseases in North America. Using a large data set of 10 coding loci and 26 isolates, the phylogeny of X. fastidiosa defined three major clades. Two of these clades correspond to the recently identified X. fastidiosa subspecies piercei (PD and some ALS isolates) and X. fastidiosa subsp. multiplex (OAK, PP, PLS, and some ALS isolates). The third clade grouped all of the OLS isolates into a genetically distinct group, named X. fastidiosa subsp. sandyi. These well-differentiated clades indicate that, historically, X. fastidiosa has been a clonal organism. Based on their synonymous-site divergence ( approximately 3%), these three clades probably originated more than 15,000 years ago, long before the introduction of the nonnative plants that characterize most infections. The sister clades of X. fastidiosa subsp. sandyi and X. fastidiosa subsp. piercei have synonymous-site evolutionary rates 2.9 times faster than X. fastidiosa subsp. multiplex, possibly due to generation time differences. Within X. fastidiosa subsp. multiplex, a low level ( approximately 0.1%) of genetic differentiation indicates the recent divergence of ALS isolates from the PP, PLS, and OAK isolates due to host plant adaptation and/or allopatry. The low level of variation within the X. fastidiosa subsp. piercei and X. fastidiosa subsp. sandyi clades, despite their antiquity, suggests strong selection, possibly driven by host plant adaptation.