ABSTRACT 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 (%bcDED) 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 %bcDED were 90.2%, 66.7%, and 40.0% for S. esuriens, S. chikisanii, and S. japonicus, respectively. %bcDED showed an increasing trend with increase in Øpc and Dpc. For DED control, it is necessary to monitor and remove small branches that were previously overlooked to prevent an increase in the population of vector beetles, although %bcDED of bark beetles emerging from small branches tends to be low.