Abstract Triploid breeding plays an important role in cultivar improvement in the genus Populus L. A novel approach for triploid production with colchicine during embryo sac development was reported recently by Wang et al. (2010). In the present investigation, female catkins of Populus pseudo-simonii × P. nigra ‘Zheyin3#’ during embryo sac development were exposed to high temperature to assess the effectiveness of high temperature for induction of triploid production. In the progeny, 45 triploids were determined by both flow cytometric analysis and somatic chromosome counting. The period 66-72 h after pollination was the most suitable for high temperature-induced triploid production during embryo sac development in the ‘Zheyin3#’. Cytological analysis showed that the frequency of eight-nucleate embryo sacs rose at an increased rate during 66-78 h after pollination, which suggested that the third mitosis during embryo sac development could be the optimal stage for high temperature-induced triploid production. The highest frequency of triploid production was 40%, which was obtained in the 44°C for 2 h treatment 72 h after pollination. In view of both triploid number and production efficiency, treatments with 41°C for 4-6 h or with 44°C for 2 h during 66-72 h after pollination were both effective for triploid induction in ‘Zheyin3#’. Statistical analysis showed that the growth of triploids and diploids was not significantly different. However, highly significant differences were observed for all leaf characteristics. Finally, the significance of high temperature treatment in Populus triploid breeding programs is discussed.