In Japan, three bark beetle species, namely Scolytus esuriens , S. chikisanii , and S. japonicus , have been recently identified as new vectors of Dutch elm disease (DED), although their detailed breeding sites and relation to DED fungi acquisition have not been revealed. We investigated the diameter of branches and a trunk (branch – trunk) at the location of the maternal tunnel (Ømt) and the pupal chamber (Øpc), the distance from the pupal chamber to the bark surface i.e. depth of the pupal chamber (Dpc) on a single Japanese elm tree, and the percentages of bark beetles carrying DED fungi (�DED) among those collected from the chamber for three species. The Ømt was 11.0–25.6 cm for S. esuriens , 8.3–23.3 cm for S. chikisanii , and 1.3–3.2 cm for S. japonicus . Therefore, three species were found to exhibit breeding site segregation based on the branch – trunk diameter. The �DED were 90.2%, 66.7%, and 40.0% for S. esuriens , S. chikisanii , and S. japonicus , respectively. �DED showed an increasing trend with ...