Transcriptome generation through RNA sequencing (RNA-seq) is a technology that can be used in the high resolution and broad dynamic range gene expression studies and in the simultaneous understanding of the genes function (Wang et al., 2009). Basically, the protein-coding genes function is inferred by the analysis of structure, function and evolution of the proteins they encode (Guo, 2013). For the characterization of unannotated proteins, can result particularly useful to undertake orthology analysis. Proteome data are important resources for having an overall genome vision but at the same time achieving a high level of accuracy in comparative studies (Andolfo et al., 2014a). To this end, we sequenced and assembled the first transcriptome of zucchini cultivar “True French,” founder of important pathogen resistant commercial varieties and to harness the full potential of such data we performed also an high-quality proteome annotation. A total of 33,966 protein sequences were predicted, functionally annotated and compared to cucumber, melon, watermelon and Arabidopsis proteomes. In addition, disease resistance (R) gene family was finely characterized and several specie-specific R-genes expansion was detected in C. pepo.