Chromosome doubling strategies are commonly used as useful tool for plant breeding. Chromosome doubling agents applied to meristem are able to penetrate to different parts of it. Using of antimitotic agents leads to chromosome doubling by acting on cells' dividing. The domain of meristem layer in the plants with doubled chromosome can be determined using cytogenetic methods. This study is conducted to determine the cytological characterization of tobacco plantlets obtained from anther culture through chromosome doubling. The haploids produced from Nicotiana tabacum L. cv. Saribaglar (2n = 48) were used as plant material. The chromosome doubling was carried out using trifluralin and acenaphthene. The chromosome counting was conducted for 937 of treated (with trifluralin and acenaphthene) and control plants. The chromosome counting of 422 treated plants with trifluralin, 185 haploid (2n = 24), 199 diploid (2n = 48), 3 tetraploid (2n = 96), 8 mixedploid (haploid and diploid series) and 27 aneuploid plantlets were observed. In acenaphthene applied plantlets (249), 225 haploid, 15 diploid and 9 aneuploid plantlets were determined. Also, for determining treated plantlets ploidy level, the guard cell lengths were used. Taking into consideration the stomata length, examined total plantlets (956) were classified into three groups as haploid (572), diploid (383) and tetraploid (1).