Phytohormones are chemical compounds found naturally in plants that have a significant effect on their growth and development. The increase in research on the occurrence of mammalian sex hormones (MSHs) in plants has prompted the need to investigate the functions performed by these hormones in plant biology. In the present study, we investigated the effects of MSHs on DNA damage and DNA methylation of wheat (Triticum aestivum L.) during the seedling growth stage, using the CRED-iPBS (coupled restriction enzyme digestion/inter primer binding site) assay and iPBS analysis to determine DNA methylation status. Exogenous treatment with four MSHs (17-& beta;-estradiol, estrogen, progesterone, and testosterone) was carried out at four different concentrations (0, 0.05, 0.5, and 5 & mu;M). The highest genomic template stability (GTS) value (80%) was observed for 5 & mu;M 17-& beta;-estradiol, 0.5 & mu;M testosterone, and 0.05 & mu;M estrogen, while the lowest value (70.7%) was observed for 5 & mu;M progesterone and 0.5 & mu;M estrogen. The results of the CRED-iPBS analysis conducted on MspI indicate that the 0.05 & mu;M estrogen-treated group had the highest polymorphism value of 40%, while the 5 & mu;M progesterone-treated group had the lowest value of 20%. For HpaII, treatment with 0.5 & mu;M 17-& beta;-estradiol had the highest polymorphism value of 33.3%, while the group treated with 0.05 & mu;M 17-& beta;-estradiol and 0.05 & mu;M progesterone had the lowest value of 19.4%. In conclusion, MSH treatments altered the stability of the genomic template of wheat plants and affected the cytosine methylation status at the seedling growth stage. Upon comprehensive examination of the results, it was seen that the employed methodology successfully detected alterations in cytosine methylation of genomic DNA (gDNA), as well as changes in the pattern of genomic instability.