During the winter agricultural season of 2022-2023, a field experiment was carried out in the Al-Basatin Al-Sharqiya, located 4 km away from the center of Al-Muthanna governorate. The objective of this study was to investigate various anatomical and genetic parameters of wheat varieties under the influence of the mutagenic alkaloid colchicine at concentrations of 0, 250, 500, and 750 mg L-1. The study employed a split plot design with three replications. The main plots consisted of different concentrations of the colchicine compound, while the secondary plots comprised four varieties (Ibaa99, Mawadah, Bohuth 22, Babel). The statistical analysis yielded significant variations in the anatomical and molecular traits among the cultivars. Notably, the cultivar Mawada demonstrated superior performance in terms of stoma density on the upper leaf surface, with an average of 118.15 stomata mm-2. Conversely, the Babel variety exhibited the highest average stoma density of 113.70 stomata mm-2 on the lower leaf surface. Regarding Bohuth 22, it demonstrated notable proficiency in terms of stoma length, exhibiting an average measurement of 3.82 μm for both the upper and lower leaf surfaces. Additionally, it displayed the highest average stoma width for the lower surface, measuring at 2.66 μm. The highest mean stomatal density for both the upper and lower leaf surfaces (114.1, 104.44 stomata mm-2) was observed at a colchicine concentration of 750 mg L-1. Regarding the intersection of the treatments, it was observed that treatment C1V3 exhibited superior performance in terms of the dimensions of the stoma, specifically the length and width on the lower surface of the leaf. The average difference between the measurements was found to be 4.40-2.96 μm, consecutively. The C2V2 treatment demonstrated superior performance in relation to the stomatal density observed on the upper surface of the leaf, exhibiting an average of 132.6 stomata mm-2. The treatment labelled as C2V4 exhibited the highest average stomata density (131.85 stomata mm-2) on the lower surface of the leaf. The molecular analysis conducted using RAPD-PCR technology revealed that the primer OPA 9 exhibited a total of 37 bands. Among these bands, 6 were found to be unique, while the remaining 31 bands displayed divergence. The OPA12 primer exhibits a minimum total of 29 bands, consisting of 24 distinct bands and 5 unique bands. Notably, all primers demonstrate a 100% occurrence of differentiated bands.