Background: A significant variation in RNA interference (RNAi) efficiency hinders further functional gene studies and pest control application in many insects. The available double-stranded RNA (dsRNA) molecules introduced into the target cells are regarded as the crucial factor for efficient RNAi response. However, numerous studies have only focused on dsRNA stability in vivo; it is uncertain whether different dsRNA storage conditions in vitro play a role in variable RNAi efficiency among insects.Results: A marker gene cardinal, which leads to white eyes when knocked-down in the red flour beetle Tribolium castaneum, was used to evaluate the effects of RNAi efficiency under different dsRNA storage conditions. We demonstrated that the dsRNA molecule is very stable under typical cryopreservation temperatures (−80 and −20 °C) within 180 days, and RNAi efficiency shows no significant differences under either low temperature. Unexpectedly, while dsRNA molecules were treated with multiple freeze-thaw cycles up to 50 times between −80/−20 °C and room temperature, we discovered that dsRNA integrity and RNAi efficiency were comparable with fresh dsRNA. Finally, when the stability of dsRNA was further measured under refrigerated storage conditions (4 °C), we surprisingly found that dsRNA is still stable within 180 days and can induce an efficient RNAi response as that of initial dsRNA. Conclusion:Our results indicate that dsRNA is extraordinarily stable under various temperature storage conditions that did not significantly impact RNAi efficiency in vivo insects.