Abstract: In this study, we measured the predawn maximum photochemical efficiency of photosystem Ⅱ (F_v/F_m) in the leaves of 60 sugarcane breeding materials during an extreme low-temperature period. We also measured leaf anatomical structure, specific leaf area (SLA), and leaf resistance to punch (F_p), and analyzed the correlation between structural traits and cold tolerance. Results showed that during the coldest period in winter, 55 of the 60 samples showed cold damage (F_v/F_m < 0.7), including 35 cultivars with F_v/F_m < 0.6. F_v/F_m was negatively correlated with the lowest temperature in the coldest month and average temperature in the cold season of the source site of the breeding material. F_v/F_m was positively correlated with mesophyll thickness (MT) and leaf resistance to punch (F_p). We found that sugarcane cultivars with stronger cold tolerance had larger MT and F_p. Thus, F_p could be used as an important index for screening cold tolerance in sugarcane cultivars.