Abstract: In this research, in order to establish a FCM system in Quercus, four common nuclear isolation buffers(Tris-MgCl₂, Galbraith’s, Marie’s and Otto’s)were used to prepare samples from mature leaf tissues of four wild oaks(Quercus acutissim, Q. variabilis, Q. fabri, Q. glandulifera var. brevipetiolata). Then all samples were tested on flow cytometer. No DNA peak was detected in the samples treated with Tris-MgCl₂ and Galbraith’s buffers. Samples prepared with Otto’s buffer, except for Q. fabri, showed poor results. The coefficients of variation(CV)of the DNA peak of the 4 samples prepared with Marie’s buffer were larger than 10%. An orthogonal design was used to optimize conventional Marie’s buffer in two factors: β-mercaptoethanol and Tween 20. Results indicated that the most suitable concentration of β-mercaptoethanol and Tween 20 on Quercus acutissim, Q. variabilis, Q. fabri, Q. glandulifera var. brevipetiolata respectively was 45 mmol/L,2.5%; 15 mmol/L, 2.0%; 60 mmol/L, 1.0%; 30 mmol/L, 1.0%. CVs for Quercus DNA peak found had decreased obviously to 3.52%-4.81% by applying modified Marie’s buffers. It proved that the test results of the four Quercus samples prepared with modified Marie’s buffers were accurate and dependable. This research laid a foundation for a wide range of studies on field population of Quercus by FCM.