Catharanthus roseus is widely cultivated around the world.  Cold atmosphere plasma (CAP) has been used to improve crop plants seed germination, and plant growth. In the present study, three different times of CAP (40, 50 and 60s) were used for the treatment of C. roseus seeds, and the changes in morphological traits, antioxidant enzymes and the genetic content of the treated plants were examined. Cold plasma (50s) markedly raised the seed germination, length of the leaves and the stem length of the plants in comparison with those in the control group. The catalase and peroxidase enzyme activities had the highest value in 60 and 40 s CAP treated plants, respectively. The sequence related amplified polymorphism (SRAP) markers showed the highest degree of genetic diversity in 50s cold plasma treated plants (Ne = 1.388, I = 0.316, He = 0.217, uHe = 0.237 and P % = 50.08). Based on Neighbor Joining, principle coordination analysis (PCoA) and analysis of molecular variance (AMOVA) test, four significantly distant groups were formed. The 40s and 50s cold plasma treated plants stand far from the control plants due to genetic difference. The results indicate that cold atmosphere plasma could be used as an economic and environmentally safe tool in increasing C. roseus growth characteristics in addition to inducing genetic variations.