In this study, the mutagenic effects of different doses and exposure times of oryzalin and Nitrogen Protoxide (N2O) were tested for stimulating polyploid on 41 B and Fercal grapevine rootstocks seedlings. Ploidy changes were examined by morphological, cytological, macroscopic, and microscopic methods. Leaf thickness, chlorophyll contents, stomatal sizes, and chloroplast numbers of polyploid seedlings stimulated with mutagens increased but their stomatal densities decreased. Flow cytometry (FC) analyses were performed on 50 samples selected by morphological and microscopic preliminary determinations. In FC analyses, 1 tetraploid seedling and 4 mixoploid seedlings from Fercal offspring and 1 mixoploid seedling from 41 B offspring were verified. The nuclear DNA content of tetraploid and mixoploid seedlings were increased by 2.00 and 1.34-fold, respectively, when compared to their diploid parents. Chromosome counts in root tip samples propagated in vitro from the tetraploid Fercal offspring confirmed a 2-fold increase compared to the diploid parent. In polyploidy induction studies, it was deemed appropriate to use FC analysis and chromosome count together to confirm the ploidy levels of mutants. Oryzalin and N2O applications at different doses and exposure times were found to be effective for inducing polyploidy in 41 B and Fercal grapevine rootstocks.