Vitis shuttleworthii is one of the most disease and pest resistant grape species originated and distributed in the southeast United States. It is highly resistant to Pierce's disease, anthracnose, black rot and downy mildew diseases, which are the limited factors to grow Vitis vinifera grapes in this region. A V. shuttleworthii cDNA library was constructed with mRNA isolated from leaves and flowers harvested during anthesis. 12,008 clean EST sequences were obtained and assembled. and generated 5776 unigenes 2106 contigs and 3670 singletons). ESTs distribution based on protein function using a modified MIPS MATDB Arabidopsis Scheme revealed that 7% of the V. shuttleworthii ESTs were related to disease/pest defense or stress tolerance genes. Over 300 contigs containing complete or 90% open coding region of known functional genes were obtained. The ESTs that were annotated as pathogenesis-related proteins, enzymes in salicylic acid, jasmonic acid, and ethylene signaling, were selected for further study in order to elucidate the role and interaction of them in the signal transduction cascade that leads to grape defense gene activation upon treatment of bacterial pathogen. We report the identification of novel disease resistant genes based on preliminary pathogenesis-relative pathways network analysis.