Identifying the causative agent of a disease provides key information for diagnosis and management. Here, we identified the causative agent of a leaf spot disease prevalent in water spinach (Ipomoea aquatica) in Fujian Province, China. Ninety bacterial isolates were recovered from symptomatic leaves of water spinach collected across six growing areas in Fujian Province. Completion of Koch’s postulates confirmed that 60 yellow mucoid isolates were the causative agents of the disease. PCR amplification of 16S ribosomal RNA gene sequences demonstrated that the isolates belonged to the genus Xanthomonas. We then PCR-amplified and sequenced the recA, dnaK, gyrB, and rpoD genes of the isolated pathogens for multilocus sequence analysis. In the resulting phylogenetic tree, the isolated pathogens grouped with X. euvesicatoria pv. perforans strain TC2-1. Biological and chemical tests performed on 60 isolates showed that all of them were positive in hydrogen sulfide, litmus milk, starch hydrolysis and bile-esculin tests, but negative in nitrate reduction, pectinase activity, and oxidase activity tests. The isolates could also cause disease symptoms on tomato (Solanum lycopersicum) leaves and stems but not on pepper (Capsicum annuum) plants. Based on DNA characteristics, physiological and chemical properties, and pathogenicity test results, we propose that the leaf spot disease in water spinach is caused by X. euvesicatoria pv. perforans.