Haploids and doubled haploids plants are important materials for production of genetically homozygous lines, genetic analysis, induction of mutation, genome mapping etc. Among the haploid plants production methods, isolated microspore culture has advantages over other methods. The efficiency of microspore culture is influenced by different factors. In this research, the effects of 3 factors including cultivar (in 2 levels: Moghan1 and Atrak), pretreatment (in 4 levels: control, 21 days cold, 7 days cold+ mannitol and cold+ (chemical+heat)) and embryo induction medium (in 5 levels: NPB-99, C17, W14, CHB-2 and P2) in isolated microspore culture of hexaploid wheat were investigated. Results showed significant interaction effects between cultivars and pretreatment and embryo induction medium. In both of the studied cultivars (Moghan1 and Atrak), “7 days cold+mannitol” pretreatment produced the highest mean of embryogenesis (81.33±4.66 & 92.33±2.33), total plant regeneration (29.33±2.02 & 39.33±0.33) and green plant regeneration (12.46±0.68 & 27.03±1.35). In the case of medium, in cultivar Moghan1 the highest means of embryogenesis (422.08±7), total plant regeneration (31.66±1.45), and green plant regeneration (26.5±2.29) were produced using C17 medium, but cultivar Atrack produced the highest means of embryogenesis (211.75±8.77) and green plant regeneration (32.53±0.76) in CHB-2 medium, and the highest mean of total plant regeneration (51±3.78) in W14 medium.