Transcriptome sequencing has become the main methodology for analyzing the relationship between genes and characteristics of interests, particularly those associated with diseases and economic traits. Because of its role of functional food for humans, commercial royal jelly (RJ) and its production are major research focuses in the field of apiculture. Multiple lines of evidence have demonstrated that many factors affect RJ output by activating or inhibiting various target genes and signaling pathways. Available coding sequences from the Honey Bee Genome Sequencing Consortium have permitted a pathway-based approach for investigating the development of the hypopharyngeal glands (HGs). In the present study, 3573941, 3562730, 3551541, 3524453, and 3615558 clean reads were obtained from the HGs of five full-sister honey bee samples using Solexa RNA sequencing technology. These reads were then assembled into 18378, 17785, 17065, 17105, and 17995 unigenes, respectively, and aligned to the DFCI Honey Bee Gene Index database. The differentially expressed genes (DEGs) data were also correlated with detailed morphological data for HGs acini.