The present study described a combining thermotherapy with meristem culture for improved eradication of onion yellow dwarf virus (OYDV) and shallot latent virus (SLV) from co‐infected in vitro‐cultured shallot shoots. In vitro‐cultured shoots infected with OYDV and SLV were thermo‐treated at a constant temperature of 36°C for 0, 2 and 4 weeks, and then meristems (0.5 mm) containing 1–2 leaf primordia were excised and cultured for shoot regrowth. Meristem culture without thermotherapy produced much higher levels of survival (100%) and shoot regrowth (55%) than those (62% survival and 32% shoot regrowth) produced by the procedure combining 4 weeks of thermotherapy with meristem culture. However, much higher virus‐free frequencies (70% for OYSV, 80% for SLV and 50% for both viruses) were obtained in the latter than those (10% for OYSV, 15% for SLV and 10% for both viruses) obtained in the former. Histological and subcellular studies showed that thermotherapy imposed stress or damage to the cells of meristems, thus resulting in reduced meristem survival and shoot regrowth. Studies on virus location revealed considerable alternations of virus distribution patterns in the thermo‐treated meristems. The results of histological and subcellular studies and analysis of virus distribution pattern added valuable experimental data in the combining thermotherapy with meristem culture for virus eradication. These data provided explanations as to why combining thermotherapy with meristem culture improved the eradication of OYDV and SLV from the virus‐infected in vitro shallot shoots.