Water deficit strongly affects plant yield and quality. However, plants can minimize drought injury by adaptation mechanisms that have evolved to escape harmful conditions. The response to water deprivation is a complex trait controlled by several genes. In order to gain a deeper understanding of drought response mechanisms in tomato, a collection of 27 genotypes was studied under different water deficit conditions. Since developmental stages might be differently influenced by drought, analyses were carried out on young plantlets during fruit setting. The only genotype that showed good performances both as water retention and fruit production was the ecotype Siccagno. All the genotypes were analyzed at molecular level with the aim of detecting structural polymorphisms in selected stress-responsive genes. In addition, the expression level of a number of these genes was measured in the genotypes more tolerant to water deficit. Many polymorphisms were detected in six stress-responsive genes, and some could imply significant modifications in the protein structure. Furthermore, the expression analysis by RT-qPCR of three stress-responsive genes allowed arguing that a higher level of expression of the gene erd15 might be related to the better response to water deficit exhibited by Siccagno. Similarly, the lower expression of eight genes in the same genotype analysed through a microarray experiment confirmed the involvement of these stress-related genes in the tomato response to drought. Further investigations are required for a better comprehension of the mechanisms underlying response to water deficit in tomato by exploiting the genetic resource identified as more tolerant. The use of new technologies able to globally analyze structural polymorphism and expression level of genes will succeed to identify crucial genes involved in stress response in the ecotype Siccagno grown under different water regimes.