Apple stem grooving virus (ASGV) and Apple chlorotic leaf spot virus (ACLSV) are two important viruses in pear, which are worldwide distributed.In commercially cultivated pear, these two viruses usually do not cause visible symptoms, but they decrease the growth and productivity of infected trees.One of most effective measures for the controlling of these virus diseases is utilizing certified healthy propagation materials.Thermotherapy was the most widely used measure for the production of virus-free plant materials.In this study, for obtaining higher virus elimination efficiency, the distribution features of Apple stem grooving virus (ASGV) and Apple chlorotic leaf spot virus (ACLSV) in in vitro-cultured pear plants was investigated by using in situ tissue-printing hybridization (TPH) and tissue blotting immunoassay (TBIA) to detect viral RNAs and coating proteins.Both ASGV and ACLSV showed high concentrations in the tip of the pear plants and lower concentrations in the middle stem.The highest viral RNA titers were found in the phloem parenchyma of vascular bundles.Monitoring of viral RNA concentrations was conducted on infected in vitro-cultured pear plants during heat-treated periods of 50 days at 34℃/42℃ (day/night regime of 16 h light at 42℃, and 8 h dark at 34℃) by using TPH combined with X-ray film exposure in serial cross sections.No viral RNA was detected in tips of 2 mm and 0.5 mm long.The heat treatment was less effective to reduce virus titers in the bottom shoot.The distribution of ACLSV and ASGV at viral RNAs and proteins levels in the whole in vitro-cultured pear shoots before and after thermotherapy was analyzed.The result would assist in the selection of in vitro-cultured plant parts with low virus concentration to be used for thermotherapy, and improving the selection meristem tips of proper sizes of in vitro cultured pear shoots for sub-culturing thus ensuring complete elimination of ACLSV and ASGV efficiently for micro-propagation.