To explore the stability of insect resistance during the development of transgenic insect-resistant trees, this study investigated how insect resistance changes as transgenic trees age. We selected 19 transgenic insect-resistant triploidPopulus tomentosalines as plant material. The presence of exogenous genes and Cry1Ac protein expression were verified using polymerase chain reaction (PCR) and enzyme-linked immunosorbent assay (ELISA) analyses. The toxicity forClostera anachoretaandLymantria disparwas evaluated by feeding fresh leaves to first instar larvae after the trees were planted in the field for 2 years and after the sixth year. Results of PCR showed that the exogenous genes had a long-term presence in the poplar genome. ELISA analyses showed significant differences existed on the 6-year-old transgenic lines. The insect-feeding experiment demonstrated significant differences in the mortality rates ofC. anachoretaandL. disparamong different transgenic lines. The average corrected mortality rates ofC. anachoretaandL. disparranged from 5.6-98.7% to 35.4-7.2% respectively. The larval mortality rates differed significantly between the lines at different ages. Up to 52.6% of 1-year-old transgenic lines and 42.1% of 2-year-old transgenic lines causedC. anachoretalarval mortality rates to exceed 80%, whereas only 26.3% of the 6-year-old transgenic lines. The mortality rates ofL. disparexhibited the same trend: 89.5% of 1-year-old transgenic lines and 84.2% of 2-year-old transgenic lines causedL. disparlarval mortality rates to exceed 80%; this number decreased to 63.2% for the 6-year-old plants. The proportion of 6-year-old trees with over 80% larval mortality rates was clearly lower than that of the younger trees. The death distribution ofC. anachoretain different developmental stages also showed the larvae that fed on the leaves of 1-year-old trees were killed mostly during L1and L2stages, whereas the proportion of larvae that died in L3and L4stages was significantly increased when fed on leaves of 6-year-old trees. Results of correlation analysis showed there was a significant correlation between the larvae mortality rates of trees at different ages, as well as between Cry1Ac protein contents and larvae mortality rates of 6-year-old trees.