Tissue culture could be considered as one of the stress factors that affect the activation of retrotransposons. In this study, mature barley embryos (Hordeum vulgare L., cv. Tokak 157/37) were cultured for callus formation. Sukkula (a non-autonomous retrotransposon) polymorphism was investigated in calli with different culturing time (40- and 80-day-old), which was derived from the same embryo in MS medium supplemented with 3 mg/L of Dicamba. Polymorphism was detected using Inter-Retrotransposon Amplified Polymorphism (IRAP) and Inter-Primer Binding Site Amplification (iPBS) techniques with two different Sukkula-specific primers. Noncultured five mature embryos were used as a control. While up to 14% polymorphism rate was observed in the case of noncultured embryos, 61 and 0% polymorphism rates were observed with IRAP analyses for 40- and 80-day-old calli, respectively. iPBS analysis revealed the polymorphism ratio up to 29% in noncultured material, and up to 58 and 70% in 40- and 80-day-old callus materials, respectively. In addition, there were 50 and 74% polymorphism rates between 40- and 80-day-old calli by IRAP and iPBS analyses, respectively. The results obtained showed that tissue culture conditions and callus age affected Sukkula retrotransposon movements, and all individuals did not present the same effect.