Lilium brownii var. colchesteri has unique and ornamental floral characteristics in graceful harmony with flower and anther colors, flower shape, elegant fragrance, and flower color change from yellowish cream to white during anthesis. There are, however, few accessions conserved in Japan up to the present, and they often show abnormally shaped flowers and mosaic leaves seemingly due to virus infection. Virus-free bulblets were established by combining meristematic tip culture and chemotherapy. At initial diagnosis with RT-PCR, Cucumber mosaic virus (CMV), Lily mottle virus (LMoV) and Lily symptomless virus (LSV) were detected from leaf tissues of the mother plants. All regenerated bulblets obtained by meristematic tip culture of the mother plants were still infected by at least one of the viruses. The meristematic tip of the bulblets infected with either LSV or LMoV was selected for subsequent culture with 2,4-dioxohexahydro-1,3,5-triazine (DHT), an antiviral chemical. LSV was eliminated successfully in mericlones from bulblets with LSV, whereas LMoV was not from those with LMoV. The virus-free bulblets were transferred to new medium without DHT, and multiplied by in vitro scaling. They were then acclimated in a phytotron glass room at 20°C. The plants were confirmed to be virus-free after 18-months’ acclimation. It was concluded that the combination of meristem tip culture and chemotherapy is practical for producing virus-free plants of L. brownii var. colchesteri.