Ginger ( Roscoe), a perennial herb, is one of the most economically valuable plants in the Zingiberaceae family. Gingerol, as the major constituents of ginger essential oil, contributes to the unique flavor and pharmaceutical value of ginger. However, the pathway of gingerol biosynthesis has not been verified and described in ginger to help understand the biosynthesis of secondary metabolites in nonmodel species. In this study, the concentrations of gingerols were quantified at different stages of rhizome development and in different tissues. The results confirmed that rhizomes are the major source of gingerols and that accumulation of gingerols in the rhizome starts at an early developmental stage. We also assembled a reference ginger transcriptome, which is composed of 219,479 unigenes consisting of 330,568 transcripts and provides a high-quality genetic resource for further research. An analysis of differentially expressed genes (DEGs) identified 12,935 DEGs among several different comparisons. Five genes [ (), (), p (), O (), and ()] associated with gingerol biosynthesis were identified as being significantly differentially expressed in the rhizome at an early developmental stage and all five genes were upregulated. Expression analysis revealed that different loci of these genes have become functionally specialized in different tissues and different developmental stages of the rhizome (subfunctionalization). Among the DEGs, and may act as gatekeepers and rate-limiting enzymes in the gingerol biosynthesis pathway and thus play an important role in regulating the biosynthesis of gingerol.