Tissue culture is a vital aspect of plant biotechnology that facilitates the development of diseaseresistant and stress-tolerant crops that are challenging to produce by conventional breeding methods. The present study evaluated the callus induction and regeneration capability of five high-yielding wheat varieties by optimizing the concentrations of different plant hormones. Immature embryos from five wheat varieties (Akbar-2019, Arooj-2022, Dilkash-2020, Subhani-2021, and MH-2021) were used in the experiment. Three plant hormones, i.e., 2,4-D (2,4-Dichlorophenoxyacetic acid), IBA (indole-3–butyric acid), and NAA (naphthalene acetic acid), each having 5 levels (1 to 5 mg/L), were tested for devising an efficient procedure for callus induction. Callus tissues were subsequently subjected to regeneration using 3 levels of kinetin (0.5, 1, and 1.5 mg/L) and 2 levels of NAA (0.5 and 1 mg/L). Callus formation was absent at all levels of NAA and IBA but was observed at all levels of 2,4-D except at 1 mg/L. The most effective concentration for callus induction across all varieties, except Subhani, was 3 mg/L of 2,4-D. Shoot regeneration was at its maximum at 1.5 mg/L across all wheat varieties, while root regeneration was observed at both levels of NAA, with the most prominent at 1 mg/L. Among the varieties tested, Dilkash, MH, and Arooj showed superior regeneration potential, while all varieties except Subhani demonstrated strong callogenesis potential. These findings suggest that a protocol utilizing 3 mg/L 2,4-D, 1.5 mg/L kinetin, and 1 mg/L NAA is optimal for the propagation of these wheat varieties. This research advances tissue culture techniques for wheat by optimizing hormone concentrations, leading to the formulation of an efficient and reliable protocol for wheat culturing. It highlights the varietal differences in tissue culture responses, aiding breeders in propagating disease-resistant and high-yielding wheat varieties.