Grapevine, Vitis vinifera L., is an important crop species of the Vitis genus that includes two subspecies, the cultivated form Vitis vinifera subsp. vinifera and the wild form Vitis vinifera subsp. sylvestris. One of the most distinguished characteristic between cultivated and wild grapevine is the sexual system. Flowers of cultivated plants are hermaphroditic, whereas wild plants have female and male flowers in separated individuals. Both the female and male flowers follow a hermaphroditic development pattern during early stages of floral development. Unisexuality arises by organ abortion at a late stage of flower development. The development of unisexual flowers from an originally bisexual floral meristem suggests that a genetic mechanism involving the arrest of stamen or pistil development must become active. The main goal of this work was to gain insights into the molecular mechanisms of sex specification in Vitis. The sequencing of the transcriptome of different stages of male, female and hermaphrodite flower buds was performed using Illumina RNA-Seq technology. The temporal expression pattern of the Vitis homologues of genes integrated in the flower organ identity model ABCDE, was analyzed in female, male and hermaphrodite flowers of grapevine. In addition, the spatial expression pattern of the B and C class genes was compared between the female and male flowers of Vitis sylvestris. The results suggest that the molecular pathway leading to unisexuality in grapevine may act after floral organ identity has been established. The comparison of the transcriptomic data of different development stages of the three flower types allowed the identification of candidate genes that might play an important role in the arrest of the rudimentary flower organs as well as in the development of the reproductive organs. Finally, a Single Nucleotide Polymorphisms (SNP) search was initiated using transcriptome data of the female, male and hermaphrodite Illumina sequenced-plants in order to find molecular markers for each type of plant sex in Vitis vinifera L. This study generated a set of tools that will help uncover the genetic molecular mechanisms involved in the development of grapevine unisexual flowers.