Polyploidy has been used in horticulture as a breeding tool to enhance characteristics such as flower size, flower number, plant size, increased width-to-length ratio of leaves and leaf thickness. Nitrous oxide gas (N2O) was applied to seedlings and zygotes of many plants as a polyploidizing agent instead of colchicine treatments. N2O application to obtain polyploid grapevines has not been tested before and has been used as a new approach in viticulture. In this study, N2O was applied in order to induce mitotic ploidy of 41B grapevine rootstock and ‘Trakya İlkeren’, ‘Gök Üzüm’ and ‘Ekşi Kara’ grape cultivars (Vitis vinifera L.) single node cuttings. The application of N2O was performed for 24 h at 12000 kPa and 25°C, after the cuttings sprouted and hand 2-3 leaf developed at different stages of mitosis. The effects were evaluated by stomata traits (density, length, width, and area) and by flow cytometry (FC) analysis using a high-performance multi-laser system. The results showed that stomatas were morphological changed by N2O applications at a significant level. The stomata density was significantly decreased (-28%) in 41B rootstock; grapevine cultivars were also significantly affected. On the other hand, the FC analysis did not detect any polyploid induced plants. The present study reveals that polyploid frequency obtained using N2O was low in the tested rootstock and grapevine varieties; this was likely is due either to the sensitivity of the diploid rootstock and grape cultivars genotypes or to the N2Otreatment method.