In the current study; mature bread wheat (Triticum aestivum L.) embryos from doubled haploid wheat line obtained from Pehlivan cultivar were cultured on drought stress induction medium (MS + 0.1 mgL-1 2.4-D + diffe- rent concentrations PEG 6000 (0, 10, 20, 40 and 60 gL-1) for 28 days. Effects of drought stress on regenerated plants and methylation alterations due to both drought stress treatment and in vitro culture conditions were investigated in the combination of Inter-Retrotransposon Amplified Polymorphism (IRAP) with methylation sensitive restriction enzyme (MspI and HpaII) digestions techniques on 28-day-old control and stress treated regenerated plants. Drought stress decreased the average of plant height and fresh weight of regenerated plants than the controls. A 90% polymorphism rate and 0.6 epigenetic similarity were obtained between cont- rols and drought stress treated experimental groups with the combination of methylation-sensitive restriction enzymes and IRAP. Additionally, the epigenetic similarity ratios between the two controls were detected to 0.733. As a result of these; to investigate methylation alterations due to both in vitro culture conditions and drought stress applications under in vitro conditions can be obtained more realistically performance with using genetically pure line.