Nonrecombinant strains of Xylella fastidiosa subsp. multiplex (those lacking evidence of significant intersubspecific homologous recombination) infect the xylem of a wide range of native and nonnative trees in North America. However, the degree to which different strains have a specialized host range remains poorly understood. We tested eight strains isolated from five different tree species (almond, olive, sweetgum, and plum in California and oak in Washington, DC). Experiments were conducted in greenhouses in Riverside, CA, and each strain was tested on 11 to 15 of the 17 plant species tested. Hosts infected by the most strains were plum (5 of 8 strains) and almond (4 of 8), while their congener peach was only infected by 1 of 8. No strains infected oleander or mulberry. All strains successfully infected their original host, with peach, olive (1 of 7), and sweetgum (2 of 6) only infected by such strains. Of the 90 total strain-novel-host combinations tested, 11 resulted in unambiguous infection, 2 gave ambiguous results, and the remaining 77 failed to result in symptoms or bacterial spread. All eight strains had a unique host range, including two pairs of strains with the same multilocus sequence typing sequence type, providing strong evidence of extensive plant-host specialization. There was little evidence that host relatedness was driving host specificity.