In recent years, extreme weather events have become increasingly frequent, and low winter temperatures have had a significant impact on peach cultivation. The selection of cold-resistant peach varieties is an effective solution to mitigate freezing damage. To comprehensively and accurately evaluate the cold resistance of peaches and screen for high cold resistance among Gansu local resources, nine different types of peach were selected as test resources to assess physiological, biochemical, and anatomical indices. Subsequently, 28 peach germplasms were evaluated using relevant indices. The semi-lethal temperature (LT50) was calculated by fitting the change curve of the electrolyte leakage index (ELI) with the Logistic equation; this can be used as an important index for identifying and evaluating the cold resistance of peach trees. The LT50 values ranged from −28.22 °C to −17.22 °C among the 28 tested resources; Dingjiaba Liguang Tao exhibited the lowest LT50 value at −28.22 °C, indicating its high level of cold resistance. The LT50 was positively correlated with the ELI and malondialdehyde (MDA) content with correlation coefficients of 0.894 and 0.863, respectively, while it was negatively correlated with the soluble sugar (SS), soluble protein (SP), and free proline (Pro) contents with correlation coefficients of −0.894, −0.721, and −0.863, respectively. The thicknesses of the xylem, cork layer, cork layer ratio (CLR) and thickness/cortex thickness (X/C) showed negative correlations (−0.694, −0.741, −0.822, −0.814, respectively). Finally, the membership function method was used to evaluate cold resistance based on the ELI, MDA, Pro, SP, SS, CLR, and xylem thickness/cortex thickness (X/C) indices. The average membership degree among all tested resources ranged from 0.17 to 0.61. Dingjiaba Liguang Tao emerged prominently in terms of high-cold-resistance (HR) membership value (0.61).