Simple SummaryConstant and fluctuating temperatures can affect the growth and development of insects, which will affect the accuracy of minimum postmortem interval estimation. Our study found that the development time ofS. peregrinawas prolonged under fluctuating temperatures. The estimation of the pupal stage has a significant effect on the estimation of PMI. We explored the feasibility of different methods for intra-puparial age estimation and found that the DEG expression profile, ATR-FTIR, and CHCs detection, combined with chemometrics, had the potential to estimate the intra-puparial age ofS. peregrinaunder constant and fluctuating temperatures.AbstractSarcophaga peregrina(Robineau-Desvoidy, 1830) has the potential to estimate the minimum postmortem interval (PMImin). Development data and intra-puparial age estimation are significant for PMIminestimation. Previous research has focused on constant temperatures, although fluctuating temperatures are a more real scenario at a crime scene. The current study examined the growth patterns ofS. peregrinaunder constant (25.75 °C) and fluctuating temperatures (18–36 °C; 22–30 °C). Furthermore, differentially expressed genes, attenuated total reflectance Fourier-transform infrared spectroscopy, and cuticular hydrocarbons ofS. peregrinaduring the intra-puparial period were used to estimate age. The results indicated thatS. peregrinaat fluctuating temperatures took longer to develop and had a lower pupariation rate, eclosion rate, and pupal weight than the group at constant temperatures did. Moreover, we found that six DEG expression profiles and ATR-FTIR technology, CHCs detection methods, and chemometrics can potentially estimate the intra-puparial age ofS. peregrinaat both constant and fluctuating temperatures. The findings of the study support the use ofS. peregrinafor PMIminestimation and encourage the use of entomological evidence in forensic practice.