Cold atmospheric pressure plasma treatment is currently being explored as an alternative way to improve the germination and growing parameters of plant seeds. However, it is important to pay attention to the effect of plasma treatment on DNA damage of the seeds as well as detailed characteristics of plasma composition and parameters. The aim of this work was to study the DNA damage of plasma-treated pea seeds (Pisum sativumL.) and plasma parameters such as the chemical composition of plasma gaseous compounds and plasma radiation. Seeds were treated with plasma using the diffuse coplanar surface barrier discharge generated in different working gases (ambient air, nitrogen, oxygen and different mixtures of oxygen and nitrogen) at atmospheric pressure and at 60 s, 180 s and 300 s exposure times. DNA damage was studied using the single cell-gel electrophoresis called the comet assay and the plasma parameters were investigated by Fourier transform infrared spectroscopy and optical emission spectroscopy. Experiments in different ratios of oxygen and nitrogen were realized in order to understand the reaction mechanism between the ambient air plasma and the treated seeds. Based on our results, ambient air plasma appears to be the most advantageous for the plasma treatment due to no significant DNA damage because of the proper combination of plasma composition in combination with water vapor present in ambient air.