AT-hook proteins play an important role in regulation of eukaryotic gene expression, and implicated in plant development, organ formation, stress response, and hormonal signaling. In this study, our analysis revealed the presence of 32 genes encoding AT-hook proteins in the tomato genome, the distribution on the chromosome, the structure and function of proteins were analyzed. AT-hook proteins could be classified into 3 type based on phylogenetic analysis. , 13 of them belong to Type I, and other members have the similar classification with Arabidopsis thaliana, which was supported with organization of predicted conserved putative motifs in AT-hook proteins. Real-time fluorescence quantitative PCR were used to gain insights into the function of AT-hook gene family members during various stages of development and in response to abiotic stress and phytohormones. Some of the AHL genes were expressed in specific organs such as roots and flowers; 32 SlAHL genes showed distinct expression patterns in response to stresses of salt, SA, ABA, high temperature and cold, the expressions of several genes were significantly up/down regulated, implying that these members might participate in regulating the defense response against abiotic stresses. The results will provide a very useful reference for cloning and functional analysis of each member of AT-hook gene family in Solanaceae crops.