Xylella fastidiosa (Xf) causes diseases in many agricultural, horticultural, and landscape crops, including Pierce's disease (PD) of grapevines. PD has been a serious problem in scattered table, wine and raisin grape growing regions throughout California, as well as in Texas for more than 100 years. In this study, we evaluated the genetic variation in diversity and structure of Xf associated with PD of grapevines in California and Texas using simple sequence repeat (SSR) markers. The analysis revealed a high level of genetic diversity across the populations of Xf isolated from the grapevines from different viticulture locations or counties in California and Texas. Overall genetic diversity of Xf was detected comparatively high in Texas (0.835) than that in California (0.662). Hierarchical analysis of molecular variance (AMOVA) showed a significant genetic structure of Xf population between California and Texas. UPGMA clustering analysis and the Bayesian clustering algorithm using STRUCTURE consistently placed California and Texas Xf populations into two major genetic groups with no clear population structure within each state.