Carvacrol and thymol, the main components of essential oil in oregano (Origanum vulgare), have strong antibacterial activity, and have big potential to replace antibiotics. In order to acquire valuable genetic variation in oregano,60Co-γ ray radiation was utilized in breeding of oregano. The volatile components were analyzed by SPME-GC-MS, also principal component analysis and hierarchical clustering composition were performed. The effects of60Co-γ ray radiation on morphological characteristics were studied, including glandular hair density, glandular hair size and volatile components of M1 generation of oregano. The results showed that the LD50 of oregano seeds was 16.39 Gy; The plant height, stem diameter, branching number, leaf length and width of irradiated M1 generation were all changed, and several morphological mutants were selected; At the same time, the density and size of glandular hair changed in a variety of ways, and 25 mutants with significantly increase in density and size of glandular hair were selected from 163 surviving plants;60Co-γ ray treatment had little effect on volatile components, but significant effect on the content of volatile components, and 5 chemotypes in oregano were induced, including carvacrol-type, thymol-type, γ-terpinene-type, β-caryophyllene- type, and germacrene-type. Six mutants with high carvacrol and thymol were obtained. These results confirmed that60Co-γ ray radiation could be useful as an effective mutagenic method for oregano breeding, and the suitable radiation dose was 20 Gy. This study provided basic data and new way for the breeding and selection of new germplasm resources in oregano.