Abstract: Using Petroselinum crispum as the calibration standard, we used two different test processes to estimate the genome size of Chinese old garden rose. Using chromosome counting method, we also determined the ploidy level of three Chinese old garden rose cultivars, which are still in debate or have not been reported, to offer necessary support for estimating the genome size. Main results are as follows. 1) The Chinese old garden rose cultivar ‘Pingdong Yueji' is diploid (2n=2x=14), while ‘Jünang' and ‘Mudan Yueji' are tetraploid (2n=4x=28). 2) Significant differences were observed in six cultivars, but no significant differences in other twelve cultivars in both two test processes, in which the method II was more accurate and stable to estimate the genome size. Method I was easier in operating and more suitable for detecting ploidy level. 3) The genome size of 18 Chinese old garden rose cultivars (including diploid, triploid and tetraploid cultivars) ranged from 0.62pg to 0.71pg. The 2C DNA amounts of diploid cultivars ranged between 1.34-1.43pg, and C-value varied from 0.67pg to 1.43pg. The 2C DNA amounts of triploid cultivars ranged between 1.96-2.05pg, and C-value between 0.65-0.68pg. The 2C DNA amounts of tetraploid varieties varied from 2.49 to 2.63pg, and C-value from 0.62 to 0.66pg. Diploid cultivars possess the largest genome, followed by the triploid ones, and tetraploid ones the least. We selected the optimum pretreatment method for estimating the genome sizes of 18 Chinese old garden rose cultivars using flow cytometry. Our results offer theory for revealing the relationship between the origin and evolution of Chinese old garden rose, and lay a foundation for their genomic sequencing.