The plant pathogenic bacterial species Xanthomonas translucens comprises diverse strains that cause vascular and non-vascular symptoms of barley. Xanthomonas translucens pv. undulosa (Xtu) infects the non-vascular, mesophyll tissue, while X. translucens pv. translucens (Xtt) colonizes, the water-transporting xylem vasculature. It remains unknown how these pathogens modify the plant environment during infection to get host nutrients. The aim of this study was to define the barley metabolites associated with the tissue-specific host colonization. Through barley metabolic profiling, we compared the healthy apoplastic and xylem fluids against fluids from apoplast infected with Xtu and xylem infected with Xtt. We described the metabolic changes in barley tissues caused by both pathogens during colonization. This work provides a model and intellectual framework to determine the basis tissue-specific behavior of vascular and non-vascular pathogens of important crop hosts.