The ability of plants to endure environmental stress factors, which are going to be more severe due to global warming, is important especially for forest plants. Because obtain trait of resistance to temperature using conventional breeding for woody plants is a time consuming way. In this study, chloroplast-localized OsHSP26 gene was overexpressed in Populus alba L. to breed tolerant transgenic poplar to temperature stress. The plantlets of OsHSP26-overexpressed transgenic poplar showed more heathy phenotypic response than wild-type plants under both prolonged low- and high-temperature stress. While the SPAD value, which refers chlorophyll content, in wild-type plants decreased depending on the exposure time to the temperature stress, higher SPAD value were shown in the transgenic plants. The contents of total phenolic compounds in the transgenic plants were lower than those of the wild-type plants, and not significantly changed except in the treatment of prolonged low-temperature. However, the total flavonoids contents of the transgenic plants were dramatically increased under prolonged temperature stress. The DPPH scavenging activities of the transgenic plants were higher than those of the wild-type plants under temperature stress. Consequently, it was revealed that overexpressing OsHSP26 allow for P. alba to be tolerant to temperature stress.