Huanglongbing(HLB, or citrus greening) is the most destructive disease of citrus, which is associated with Candidatus Liberibacter asiaticus(Las). Few management options are available, aside from preventive measures such as removing infected plants, planting disease-free seedlings, and managing the insect vector. In this study, we assessed the efficacy of thermotherapy against HLB under controlled greenhouse conditions. A total of 60 two-year-old, graft-infected Citrus reticulata Blanco plants were used. The plants were randomly divided into three groups(45°C, 48° C, and untreated control), with five plants/replicate(rep) and four reps/treatment. The treated plants were placed in phytotrons for a 4-h treatment session, repeated once per week for three consecutive weeks. Disease remission was observed eight weeks post-treatment. Real-time PCR assays revealed that Las titers in HLB-affected seedlings were significantly reduced in both 45 and 48°C treatments four weeks after treatment, with the exception of eight plants. In contrast, Las titers in the untreated control plants increased significantly during the same period, with a maximum increase of 28-fold. Except for seven plants, Las titers in the new flushes of treated plants decreased more than 90% eight weeks after treatment. Las titers in mature leaves of treated plants decreased 56 and 60% in average at 45 and 48°C, respectively, eight weeks after treatment. The HLB symptoms and Las titer of seedings were markedly alleviated eight weeks after treatment in both 45 and 48°C treatments. Our results laid a good foundation for the further development of citrus free-disease seedling cultivation and Huanglongbing control in the field. The whole plants were replaced for scion or branch in previous as the research object in this study, and the expression of Huanglongbing symptoms combined with real-time polymerase chain reaction(PCR) were used to evaluate the effect of heat treatment in the greenhouse. Huanglongbing (HLB, or citrus greening) is the most destructive disease of citrus, which is associated with Candidatus Liberibacter asiaticus (Las). Few management options are available, aside from preventive measures such as removing infected plants, planting disease-free seedlings, and managing the insect vector. In this study, we assessed the efficacy of thermotherapy against HLB under controlled greenhouse conditions. A total of 60 two-year-old, graft-infected Citrus reticulata Blanco plants were used. The plants were randomly divided into three groups (45 ° C, 48 ° C, and untreated control) with five plants / replicate (rep) and four reps / treatment. The treated plants were placed in phytotrons for a 4-h treatment session, repeated once per week for three consecutive Real-time PCR assays revealed that Las titers in HLB-affected seedlings were significantly reduced in both 45 and 48 ° C treatments four weeks after treatment, with the exception of eight plants. In contrast, Las titers in the untreated control plants increased significantly during the same period, with a maximum increase of 28-fold. Except for seven plants, Las titers in the new flushes of treated plants decreased more than 90% eight weeks after treatment. Las titers in mature leaves of treated plants decreased 56 and 60% in average at 45 and 48 ° C, respectively, eight weeks after treatment. The HLB symptoms and Las titer of seedings were markedly alleviated eight weeks after treatment results in both 45 and 48 ° C treatments. Our results laid a good foundation for the further development of citrus free-disease seedling cultivation and Huanglongbing control in the field. The whole plants were replaced for scion or branch in previous as the research object in this study, and the expression of Huanglongbing symptoms combined with real-time polymerase chain reaction (PCR) were used to evaluate the effect of heat treatment in the greenhouse.