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Bougainvillea Comm. ex Juss. (Nyctaginaceae; Bougainvillea) is a popular ornamental plant with vigorous growth, luxuriant blooming, colorful bracts, and a high tolerance to the stresses of temperature, drought, and soil pollution, and thus is widely cultivated in tropical and subtropical regions. However, the paucity of information on ploidy and the genomic constitution is a significant challenge to genome research and cultivar improvement. We present a flow cytometry method for ploidy detection in bougainvillea based on evaluating different lysates and tissues, identify the ploidy level of a batch of bougainvillea accessions, and infer the genome size of horticultural species Bougainvillea glabra, Bougainvillea spectobilis, and Bougainvillea peruviana. The results show that tender leaves and woody plant buffer (WPB) were optimal for flow cytometry analysis. The 2C nuclear DNA amounts in 176 bougainvillea accessions ranged from 4.66 ± 0.04 to 12.26 ± 0.1 pg, which represents 161 diploids, 13 triploids, 1 tetraploid, and 1 di-tetraploid mixoploid. For B. glabra, B. spectobilis, and B. peruviana, the mean 1C values were 3.201, 3.066, and 2.915 pg, respectively. The genome size of B. glabra was significantly larger than that of B. peruviana (P = 0.0004), but had no significant variation with that of B. spectobilis (P = 0.1061). These results reveal fundamental cytogenetic information for bougainvillea that are beneficial to whole-genome sequencing and hybrid breeding programs.