To clarify the theoretical basis of the differences in high temperature stress tolerance among herbaceous peony (Paeonia lactiflora Pall.), we investigated the heat injury index of twelve P. lactiflora cultivars. Of these, heat-tolerant ‘Zifengyu’ and moderately heat-tolerant ‘Hongyanzhenghui’ were selected to study the biochemical and molecular responses to high temperature stress. ‘Zifengyu’ had reduced malondialdehyde (MDA) content, increased soluble sugar, chlorophyll (Chl) a, Chl b, Chl a b. and carotenoid contents, as well as elevated antioxidant enzymes activities, photosynthetic rate (Pn), transpiration rate (Tr) and relatively intact cellular structures compared with ‘Hongyanzhenghui’, especially when the temperature was the highest. Additionally, we isolated partial cDNAs of two heat shock protein genes (HSP60 and HSP90) from P. lactiflora, which were 880-bp and 1077-bp nucleotides in length, respectively. The expression levels of PlHSP60, PlHSP70 and PlHSP90 were lower in ‘Zifengyu’ than in ‘Hongyanzhenghui’ for the first three of four developmental stages examined. These results indicated that heat-tolerant P. lactiflora cultivar could effectively scavenge reactive oxygen species (ROS), protect cellular structures, reduce thermal damage and delay the death of plants by enhancing antioxidant enzymes activities and HSP expression under high temperature stress. These findings provide a theoretical basis for breeding heat-tolerant P. lactiflora cultivars.