Potato is the fourth most valuable plant for human nutrition. In addition to carbohydrates, potato tuber contains important vitamins and micronutrients for human health. During developmental stages of Potato; morphological, physiological and molecular changes occur. From the genetic point of view, studying these events is crucial for breeding potatoes with nutritional values and a higher yield. The next-generation sequencing methods generate abundant and useful genetic data for molecular breeding of crop plants. In this study, two different potato tuber developmental stages (S1 and S2) were chosen and sampled. Following RNA extraction and cDNA synthesis, Illumina RNA sequencing was performed in two replications for each stage. Bioinformatics analysis, gene ontology, and gene set enrichment analysis were performed. Finally, out of 1829 differential expression genes, 1186 genes were identified and valided by bioinformatics resources. The result of gene ontology comparison showed that 393, 483 and 669 genes were involved in biological processes, cellular components, and molecular functions, respectively. Most genes were present in the phenylpropanoid biosynthesis, biosynthesis of secondary metabolites and metabolic pathways. Peroxidase and membrane transporters coding genes were the most important genes at the beginning of tuber onset to tuberization. These results suggested that development in potato tuber activates metabolic pathways, which not only promotes growth and development but also activates the pathways involved in stress responses and synthesis of different compounds. Ten key genes were identified involved in starch biosynthesis, most of which showed a significant up-regulation, suggesting that the starch biosynthesis pathway starts from the onset of stolon development to tuber maturation.