Tomato (Solanum lycopersicum) stress resistance and fruit total soluble solid (TSS) content have changed dramatically during selective breeding, and transcriptome variation has played a critical role in this rewiring. However, the single tomato reference genome impedes characterization of whole-transcriptome variation during domestication and breeding at the population level. Here, we constructed a pan-transcriptome of orange-stage tomato fruit, and investigated global expression presence/absence variation (ePAV) and differentially expressed genes (DEGs) based on RNA sequencing (RNA-seq) data from 399 tomato accessions. A total of 7181 genes absent from the reference genome were identified, 6122 of which were ePAV genes during tomato domestication and breeding including resistance genes such as late blight resistance gene PIM_DN29746_c0_g3_i1 and peroxidase P7-like gene PIM_DN30274_c0_g2_i1. In addition, 3629 genes were significantly differentially expressed during tomato selection, among which 19 genes were associated with the reduced fruit TSS content of mod tomato cultivars, including LIN5, TIV1, and seven novel sugar transporter genes. Our results indicate that natural and artificial selection greatly shaped the tomato transcriptome, thereby altering the fruit TSS content and resistance to abiotic and biotic stresses.