Abstract: Tissue culture conditions for two varieties of wheat (Triticum aestivum L.), Fakhr-e-Sarhad and Gandam-2002 were optimized using mature seeds as source of explant. Both of these varieties were tested for their callus production, callus proliferation and regeneration abilities. Different concentrations of 2,4-D (2,4-Dichlorophenoxy acetic acid) were tested for callus induction. Both of these varieties showed maximum callus induction (46.87% for Fakhr-e-Sarhad and 62.5% for gandam-2002) on MS medium supplemented with 3.5 mg L-1 2,4-D. Excellent callus proliferation (87.5% for Fakhr-e-Sarhad and 95.31% for Gandam-2002) was obtained on MS medium containing 4 mg L-1 2,4-D, also callus proliferation (81.25% for Fakhr-e-Sarhad and 93.75% for Gandam-2002) was found on 3 mg L-1 BAP (Benzyl aminopurine). Both varieties were regenerated into whole plant on medium containing 1 mg L-1 IAA (Indol, 3-acetic acid) in combination with 2 mg L-1 BAP after a culture period of 3 weeks. The regeneration was 50 and 60% for Fakhr-e-Sarhad and Gandam-2002, respectively on medium containing 3 mg L-1 Kinetin. The Gandam-2002 produced an average of 4 plantlets per callus, while the Fakhr-e-Sarhad produced an average of 2 plantlets per callus on medium fortified with 1 mg L-1 IAA along with 2 mg L-1 BAP.