In a mass selection program, six interesting lines for phenotypic traits of chrysanthemum (Dendranthema × grandiflorum (Ramat.) Kitam.) for cut flowers were selected from a population of 'May Shoesmith'. After further cycles of selection, two superior plants, namely '81M' and '15M', showed interesting agronomic and commercial characteristics, among which were plant height, flower size and long-lasting flowers. According to the EU regulations, only chrysanthemum plants free from "quality pathogens" (i.e, Tomato Aspermy Virus and Chrysanthemum B Mosaic Virus) can be produced and commercialized. Hence, a protocol for in vitro thermotherapy, shoot-tip culture and shoot proliferation was optimized for the two selected lines. After the introduction in vitro, a 30-day cycle of heat temperature (from 25°C to 30°C for 12 days and at 30°C for 18 days) was developed, followed by shoot-tip culture and shoot proliferation. On average, only 10% of the new shoots were found to be virus-free by ELISA test. Rooting and plant acclimatization, for both the selections, were carryed out to obtain virus-free stock plants, used as source of high-quality propagation material which is distributed to nurseries.