Pierce’s disease (PD), caused by bacterium Xylella fastidiosa, seriously hampers the cultivation of Vitis vinifera (bunch grapes) worldwide. The bacterium clogs xylem vessels and forms a biofilm, resulting in the wilting of the plant. Vitis species, such as, Florida hybrid bunch (FH) and muscadine grape (Vitis rotundifolia) are cultivated in United States, and are known for their tolerance to PD. The overall goals of this study are to determine the signal sequences associated with xylem and sap for the delivery of therapeutic proteins to control Xylella fastidiosa. The objectives of this research project are: 1) to compare the proteome profiles of xylem tissue and xylem sap from PD tolerant and -susceptible grapevine cultivars, and 2) to determine the role of proteins in the tissue and sap associated with PD tolerance mechanism. In this study, we used Bunch, FH, and Muscadine grape cultivars to characterize differentially expressed and unique proteins using LC MS/MS spectrometry searched against Vitis database. A total of 2519 and 402 proteins were identified in xylem and sap respectively, of which 151 proteins were common to both tissues. The cluster dendrogram analysis of the sap proteome showed that all of the Vitis species are bifolious. Florida hybrid bunch and muscadines are more closely related to each other than to bunch grape. Functional analysis and gene ontology revealed that proteins involved in carbohydrate metabolic process are more abundant in bunch grape, while FH and muscadine grape have more defense related proteins. Proteins involved in the defense and peroxidase activity are abundantly present in xylem and sap of FH and muscadine, and these proteins are relatively in reduced levels in bunch xylem and sap. Together, our findings highlight the possible roles of the identified unique proteins that confer PD tolerance to Florida hybrid bunch and muscadine cultivars.