BACKGROUND: Temperature is one of the essential abiotic factors required for honey bee survival and pollination. Apart from its role as a major contributor to colony collapse disorder (CCD), it also affects honey bee physiology and behavior. Temperature-stress induces differential expression of genes related to protein synthesis and metabolic regulation, correlating with impaired gland function. This phenomenon has been confirmed in mandibular glands (MGs), but not in Hypopharyngeal glands (HGs), potentially affecting larval nutrition. RNA-seq analysis was performed using HGs tissue at low (23 °C), regular (26 °C), and high (29 °C) ambient temperatures. This study aims to decode molecular signatures and the pathways of the HGs tissue in response to temperature-stress and the rapid genetic changes that impact not only royal jelly (RJ) production potential but also other biological functions related to HGs and beyond. RESULTS: From the analyzed RNA-seq data, 1,465 significantly differentially expressed genes (DEGs) were identified across all the temperature groups. Eight genes (APD-1, LOC100577569, LOC100577883, LOC113218757, LOC408769, LOC409318, LOC412162, OBP18) were commonly expressed in all groups, while 415 (28.3%) of the total genes were exclusively expressed under temperature-stress. The DEGs were categorized into 14 functional groups and significantly enriched in response to external stimuli, response to abiotic stimuli, and protein processing in the endoplasmic reticulum (ER). Pathway analysis of exclusively temperature-stressed DEGs revealed that these genes promote ECM-receptor interaction and fatty acid metabolism while reducing protein processing in the ER, which is related to royal jelly (RJ) production and overall nutrition. Although heat-shock protein 90 and gustatory receptor 10 serve as markers for stress and hypopharyngeal glands (HGs) development respectively, their expression varies under temperature-stress conditions. CONCLUSIONS: We conclude that with the recent effects of climate change and its contributing factors, honey bee pollination, and reproduction activity is on the verge of halting or experiencing a detrimental decline. Considering the impact of temperature-stress on the expression of the nutritional marker gene (GR10), silencing GR10 in HGs tissue could provide valuable insights into its significance in nutritional performance, survival, and beyond. Finally, a broader temperature range in future experiments could help derive more definitive conclusion.