Multi-locus sequencing typing and single nucleotide polymorphisms (SNPs) were used to evaluate the molecular evolution of genes and the genetic diversity of 40 Brazilian isolates of Xylella fastidiosa (Xf) from citrus and coffee. Other five isolates from grapes, Morus rubra, or plums were added to the sample. The hypothesis that populations of Xf from citrus and coffee are not genetically subdivided was tested. SNPs were detected in 17 out of 20 randomly chosen loci of Xf. None of the molecular haplotypes of Xf was shared by different host populations. This indicates complete absence of gene flow between citrus and coffee populations of Xf. However, the reconstruction of haplotypes network by statistical parsimony supported the hypothesis of contemporaneous evolution of Xf genes from citrus and coffee.