The physiological response and differential expression of the cold resistance-related genes, Hsp90 and WRKY33, of six varieties of Magnoliaceae under different low temperatures were analyzed in this study, to determine the molecular mechanisms of cold resistance and select cold-resistant Magnoliaceae species. The results showed that the semi-lethal temperature of the six varieties of Magnoliaceae ranged from -22.06 to -10.64℃, ranging from high to low were Michelia maudiae var. rubicunda, M. wilsonii, M. martinii "Tiny" ♀×M. shiluensis, M. platypetala, M. martinii "Tiny", and Parakmeria lotungensis. During low temperatures, the indexes of soluble protein(SP), proline(Pro), superoxide dismutase(SOD), and Peroxidase(POD) initially increased first and then decreased, whereas those of soluble sugar(SS) and Malonaldehyde (MDA) continued accumulating. Relative electrical conductivity(REC), MDA, SP, SS, and Pro could be used as key indicators for the evaluation of cold resistance of six Magnoliaceae varieties. A cluster analysis of the six Magnoliaceae varieties for cold resistance was divided into strong, medium, and weak three categories, and they were P. lotungensis and M. martinii "Tiny", M. platypetala, M. martinii "Tiny" ♀×M. shiluensis and M. wilsonii, and M. maudiae var . rubicunda, respectively. Differential expression analysis revealed that the expression of HSP90 and WRKY33 genes displayed a peak followed by a progressive decline in the six Magnoliaceae varieties. At the semi-lethal temperature of each variety, the expression of both genes was strongly inhibited and irreversible as temperature decreased. There was no significant difference in the expression of both genes between different varieties at 0℃(P>0.05), but they were significantly activated at -5℃. The expression of HSP90 and WRKY33 were downregulated 0.76 and 0.68 times, respectively, than that at -5℃ in M. maudiae var. rubicunda, whereas were further activated in the other five varieties at -10℃. Under low temperature stress at -15℃, HSP90 and WRKY33 expression in the medium cold-tolerant varieties of M. platypetala, M. wilsonii, and M. martinii "Tiny" ♀×M. shiluensis was strongly inhibited, and the expression of both genes was downregulated 0.38, 0.33, 0.32, and 0.71 times, respectively, and 0.72 and 0.74 in P.lotungensis and M. martinii "Tiny", respectively. Under low temperature stress at -20℃, HSP90 and WRKY33 expression in the leaves of the six Magnoliaceae varieties were all strongly inhibited, but the expression was still higher in the strongest cold resistance variety of P. lotungensis than in the other five varieties. The activation and expression of cold-tolerant genes are important factors affecting the cold resistance of plants. Different varieties showed significantly different response mechanisms to low temperature stress. Stronger cold-tolerant varieties activated the response mechanisms more quickly to low temperature stress. Expression of cold tolerance related genes was activated to adjust the physiological and biochemical activities to resist or adapt to cold stress. However, it was inhibited in the cold-intolerant varieties and significantly decreased their adaptation to cold stress.