In order to use the electrical resistance method to accurately and timely detect and evaluate the internal decay defects of living trees, the effects of the seasonal temperature and moisture content on the electrical resistance of standing trees were investigated. At the Northeast Forestry University Experimental Forest Farm, Harbin, Heilongjiang Province of China, Populus simonii Populus simonii Carr. and Larix gmelinii (Rupr.) Rupr. were selected as the objects and the electrical resistance of standing trees was tested through different seasons from December 2016 to December 2017. Meanwhile, the effects of changes in the seasonal temperatures (−20 to −10 °C, −10 to −5 °C, −5 to 0 °C, 0 to 5 °C, 5 to 10 °C, 10 to 15 °C, 15 to 25 °C) as well as changes in the moisture content (MC) (Populus simonii, MC ≥ 103%; Larix gmelinii, MC ≥ 77.5%) on the electrical resistance in the cross-sections of living trees were studied. The influence of temperature at different moisture contents, the moisture content at different temperatures, and their combined effects on electrical resistance were analyzed, following which a regression model was also established. The obtained results indicated that ambient temperature had a significant effect on the average value of electrical resistance in the cross-section of living trees when temperatures were below the freezing point. There was a sudden discontinuity near the freezing point, and logR (logarithm value of electrical resistance) in the cross-sections of sound trees and decayed trees changed in a similar trend with variations in the temperature. While the effect of moisture content on logR in the cross-sections of threes was insignificant at different temperatures because of the moisture content above FSP (fiber saturation point). It indicated that the temperature and moisture content had interactive effects on logR in the cross-sections. The binary linear regression model between moisture content, temperature, and logR was highly fitted with a correlation coefficient (R2) higher than 0.8. The outcome of this investigation indicates that when non-destructive testing is performed on living trees using electrical resistance at different seasonal temperatures, the measured results need to consider both the temperature and moisture content. For practical work, it is not recommended to consider testing living trees near the freezing point temperature using the electrical resistive tomography. Below the freezing point, the electrical resistance changes with temperature greatly relative to the normal temperature. Therefore, when performing the detection of electrical resistance, it is necessary to calibrate the effects of temperature