Leaf scald caused byXanthomonas albilineans(Xa) is a major bacterial disease in sugarcane that represents a threat to the global sugar industry. Little is known about the population structure and genetic evolution of this pathogen. In this study, 39Xastrains were collected from 6 provinces in China. Of these strains, 15 and 24 were isolated fromSaccharumspp. hybrid andS. officinarumplants, respectively. Based on multilocus sequence analysis (MLSA), with five housekeeping genes, these strains were clustered into two distinct phylogenetic groups (I and II). Group I included 26 strains from 2 host plants,Saccharumspp. hybrid andS. officinarumcollected from 6 provinces, while Group II consisted of 13 strains fromS. officinarumplants in the Zhejiang province. Among the 39Xastrains, nucleotide sequence identities from 5 housekeeping genes were:ABC(99.6–100%),gyrB (99.3–100%),rpoD (98.4–100%),atpD (97.0–100%), andglnA (97.6–100%). These strains were clustered into six groups (A–F), based on the rep-PCR fingerprinting, using primers for ERIC2, BOX A1R, and (GTG)5. UPGMA and PCoA analyses revealed that group A had the most strains (24), followed by group C with 11 strains, while there was 1 strain each in groups B and D–F. Neutral tests showed that theXapopulation inS. officinarumhad a trend toward population expansion. Selection pressure analysis showed purification selection on five concatenated housekeeping genes from all tested strains. Significant genetic differentiation and infrequent gene flow were found between twoXapopulations hosted inSaccharumspp. hybrids andS. officinarum. Altogether, these results provide evidence of obvious genetic divergence and population structures amongXastrains from China.