Abstract: Procambarus clarkii, commonly known as “crayfish” or “freshwater crayfish”, is one of the most important freshwater aquaculture species in Chinese mainland. Recently, the aquaculture area and production of the crayfish in Chinese mainland have been continuously increased. However, the stress response caused by various factors, such as climate change, transportation and environmental change has led to more and more serious problems, including explosive disease and even death during culturing the crayfish, which not only decreased the crayfish product quality, but also resulted in serious damage to the crayfish breeding industry. In this study, we first treated the crayfish by transportation and temperature stresses to select the stronger stress resistant (SSR) and the weaker stress resistant (WSR) crayfishes. Then we dissected the hepatopancreas of SSR and WSR crayfishes to compare the metabolomics by Liquid Chromotography-Mass Spectrometry/Mass Spectrometry (LC-MS/MS). A total of 10292 ions were detected, from which 464 metabolites were identified to be significant different between SSR and WSR (fold change>1.2,P<0.05, and VIP>1.0). Among them, 227 metabolites were down-regulated and 237 metabolites were up-regulated in SSR relative to WSR. KEGG analysis showed that these differential metabolites were mainly enriched in amino acid metabolic pathways, including histidine metabolism, taurine and hypotaurine metabolism, lysine degradation, valine, leucine and isoleucine biosynthesis, and glutathione metabolism. They also enriched in ascorbate and aldarate metabolism, carbohydrate metabolic pathways (pentose and glucuronate interconversions), fatty acid metabolic pathways (unsaturated fatty acid biosynthesis), etc. These results indicate that there are a wide range of metabolic responses in response to transportation and temperature stress, and some metabolites related to antioxidative stress and enhancing immunity, such as γ-L-Glutamyl-L-cysteine, taurine, and oleic acid, may play an important role in the stress resistance inP. clarkii. This study can not only provide new insights for studying mechanisms of animal stress resistance, but also have important value in developing strategies to cope with the stress response and in breeding new aquaculture strains with strong stress resistance.