Abstract: The aim of this study is to investigate the effect of osmotic treatment on the biological characteristics of seeds of Quercus acutissima and Quercus variabilis, and the changes of free water inside seeds during osmotic treatment were analyzed, which provided a theoretical reference for the development and utilization of germplasm resources of seeding. Through the osmosis treatment by PEG-6000 at concentrations of 0%, 25%, 50% and 75%, respectively, the seeds of Q. acutissima and Q. variabilis were treated in this study, the dynamic changes of seed water content were plotted and the germination index was measured. According to differential scanning calorimetry（DSC）, the thermodynamic characteristics of seeds after different concentrations of PEG were analyzed. The ultrastructural changes of the embryonic axis cells after PEG treatment were observed by scanning electron microscopy. The results showed that the average water content（40.56%, 44.31%） and germination percent（97.77%, 95.55%） of seeds of Q. acutissima and Q. variabilis without seed coat were higher than those of seeds with seed coat（34.94%, 33.51%） and germination percent（75.00%, 77.78%）, and 50% PEG treatment had the best comprehensive effect on germination of Q. acutissima and Q. variabilis seeds, 75% PEG had the worst effect. The crystallization onset temperature, peak temperature and enthalpy of embryonic axis showed a regular decreasing trend with the increase of PEG concentration. Scanning electron microscopy showed that high concentrations of PEG led to cell degradation and some cells were wrinkled. In summary, the seed coat limits the water uptake and germination of Q. akathisia and Q. variabilis seeds. Different concentrations of PEG treatment had significant effects on the biological characteristics of seeds of Q. acutissima and Q. variabilis. The free water content of seeds decreased with the increase of PEG concentration during osmotic treatment, the cell morphological structure changed, and the seed water content and germination rate were affected, so PEG osmotic treatment could be used as a technical means for the seed treatment of Q. acutissima and Q. variabilis, and was used for the development and utilization of Q. acutissima and Q. variabilis germplasm resources.