Mulberry leaf scorch (MLS), caused by Xylella fastidiosa, is a disease of mulberry trees in the United States that has largely been documented from locations in the eastern and central areas of the country. MLS was recently detected for the first time in white mulberry (Morus alba) trees in southern California. Four MLS-strains were isolated from two locations and confirmed as X. fastidiosa by enzyme-linked immunosorbent assay (ELISA), direct isolation of the pathogen, and use of the X. fastidiosa-specific PCR primers RST31-33. Isolated strains were characterized by the sequencing of their 16S-23S rDNA intergenic spacer regions (ISR) and random amplified polymorphic DNA (RAPD) analysis and subsequent comparison with a previously characterized MLS-strain (Mulberry-VA) and representatives of X. fastidiosa subsp. fastidiosa, X. fastidiosa subsp. multiplex, and X. fastidiosa subsp. sandyi. MLS-strains isolated from California were distinct from strains causing almond leaf scorch, oleander leaf scorch, and Pierce's disease and similar to the Mulberry-VA-strain. The ISR sequences of two MLS-strains, MLS063 and MLS059, were 100% identical to that of the Mulberry-VA sequence, whereas MLS012 and MLS024 were 99.8 and 99.6% identical to the Mulberry-VA-strain and 99.4% identical among themselves. Genomic analysis using RAPD revealed no differences among the four strains. The pathogenicity of one strain, MLS063, was confirmed by inoculation of glasshouse-grown white mulberry plants. Three months after inoculation, the pathogen was recovered from 21 of 25 inoculated plants, and 5 of 25 plants were dead within a year of inoculation. Inoculation of grapevines and oleanders with MLS063 did not result in any disease or recovery of the pathogen up to 1 year later, showing that this strain was not cross-infective to these hosts.