Abstract: This study aims to conduct flow cytometry(FCM) research that can accurately determine the ploidy level, DNA content, and genome size of different Betula species. Experimental materials with different growth stages(tender leaves, young leaves, mature leaves) were treated with varying methods of storage(room temperature, cold storage and polyvinylpyrrolidone, commonly referred to as PVP). Then, FCM was used to screen and optimize 9 kinds of commonly used dissociation fluids, and the cell ploidy and DNA content of the experimental materials were detected. The results showed that fresh young leaves, soaked in 1% PVP at room temperature and the nuclear suspension prepared by MgSO4 dissociation solution, is the best dissociation fluid. Of all 7 tested Betula spp., the sequence of genomic ploidy was Betula dahurica(2.09 ± 0.04 Gb, octaploid) > Betula chinensis(1.53 ± 0.08 Gb, hexaploid) > Betula luminifera(0.87 ± 0.05 Gb, tetraploid) = Betula ermanii(0.80 ± 0.07 Gb, tetraploid) > Betula pendula(0.46 ± 0.03 Gb, diploid) = Betula nigra(0.51 ± 0.01 Gb, diploid). The ploidy level of Betula luminifera × Betula nigra hybrid progeny was found to be triploid(0.66 ± 0.02 Gb), which indicated that there was no reproductive isolation between the 2 species.