Abstract: In order to obtain materials of Nicotiana plants easily when preparing chromosome specimens, in this study, allopentaploid tobacco (2n=5x=58) was used as materials to observe the effects of ovary sizes and pretreatment methods on Nicotiana plants chromosome specimen preparation. First, ovaries were divided into 6 types based on corolla length/calyx length; and then, two pretreatments were compared, one was pretreatment in 0.002 mol/L 8-hydroxy-quinoline solution at normal atmospheric temperature for 2, 4 and 6 h, the other one was pretreatment in 0.002 mol/L 8-hydroxy-quinoline solution at 7℃ for 12, 24 and 36 h. When the length of the corolla was less than or equal to 1/2 of the calyx, the ratio of mitotic cells reached the highest, being 97.00±17.82 in 1000 cells. When ovaries with the most mitotic cells were pretreated at 7℃ for 24 h, the number and configuration of chromosomes were clear and distinguishable. The above mentioned methods were applied in chromosome specimen preparation of Yunyan87 (2n=4x=48) and N. plumbaginifolia (2n=2x=20), and nice results were obtained, respectively. In addition, 5S rDNA-FISH was carried out on chromosomes of N. plumbaginifolia, and expected recognizable signals were found on 5 different chromosomes. These results indicated that tender ovary could be used to prepare chromosome specimens of Nicotiana plants. Chromosome specimens prepared with modified method were not only beneficial to chromosomes counting and karyotype analysis, but also can be applied to in situ hybridization analysis.