A survey of urban trees affected by bacterial leaf scorch (BLS) caused by Xylella fastidiosa was conducted in the District of Columbia during 2011 and 2012. Over 20 species of urban trees were evaluated at 95 sites. Symptomatic and asymptomatic foliage from trees with BLS symptoms and foliage from neighboring asymptomatic trees were sampled. An X. fastidiosa-specific enzyme-linked immunosorbent assay (ELISA) and a polymerase chain reaction assay were used to detect and identify the strains from environmental samples. Symptomatic trees testing ELISA-positive for X. fastidiosa occurred most frequently with Quercus palustris, Q. rubra, Ulmus americana, and Platanus occidentalis. The bacterium was also less frequently identified on eight other symptomatic and five asymptomatic tree species. On infected trees, the bacterium was also detected on the asymptomatic portion of seven tree species. All strains were identified as the X. fastidiosa subsp. multiplex genotype ALSII except on Morus alba, where the genotype ALSI and the subsp. sandyi were detected. The occurrence of crown dieback was found significantly associated with X. fastidiosa-infection on Q. palustris, Q. rubra, U. americana, and P. occidentalis. Because this pathogen continues to perpetuate uncontrolled in urban environments, there is a pressing need to identify long-term management strategies that abate disease.