A total of 360 Xylella fastidiosa strains were isolated from sweet orange (Citrus sinensis') cv. Pera plants growing in five geographic regions in the Brazilian state of Sao Paulo. The genetic variation of these strains was determined by 15 variable number tandem repeat (VNTR) and 58 random amplified polymorphic DNA (RAPD) markers. The mean values of genetic diversity (H) of X. fastidiosa strains within each geographic region determined by RAPD (H-RAPD) were substantially lower than H-VNTR values. H-RAPD values ranged from 0.00 to 0.095, whereas the H-VNTR values ranged from 0.024 to 0.285. A highly significant value of Nei's coefficient of gene differentiation (G(ST)=0.355; P=0.000) was detected among all five populations. Analysis of the molecular variance (AMOVA) also revealed significant genetic differentiation among regions or populations (phi(STAT)=0.810; P<0.001). In addition, genetic differentiation among subpopulations (plants) within the regions (φ(STAT)=0.699; P<0.001) and within each plant (phi(STAT)=368; P<0.001) were statistically significant. These high values of genetic differentiation among X fastidiosa strains from different regions suggest a genetic structure according to region of host origin. However, no apparent correlation between genetic distance and region of origin of populations were supported statistically by Mantel analysis (r=0.27; P=0.22).