Abstract: In order to understand the freezing characteristics and metabolites of cassava root, the edible cassava variety “South China 9” was taken as the research object, by measuring the freezing-law and related influencing factors at the head, middle and tail of cassava root, combined with non-targeted metabolomics to analyze the differential metabolite. The results showed that: The freezing temperature was from −0.6 to −1.1 ℃ in different parts of root, but the freezing time was different. The highest values of soluble solid content and starch content in different parts appeared in the head, which were 7.00% and 26.84% respectively, but there was no significant difference among different parts (P>0.05), while the highest value of water content appeared in the tail, which was 64.07%. Correlation analysis showed that freezing point was negatively correlated with soluble solids and water content, and positively correlated with starch content. Compared with control, 41 differential metabolites were screened under low treatment by the non-targeted metabolomics, and the enrichment degree of phenylalanine metabolic pathway was the highest, with a total of 9 significant differential metabolites. In this pathway, the expression of hippuric acid and benzoic acid was up-regulated, which might be related to the response of root tubers to low temperature. The results provide theoretical reference for low-temperature storage in the future.