This paper investigated the variation law of methanol concentration in transformer oil caused by operational defects and hotspot temperature from 500 kV field transformers. The correlations between various operational defects and methanol in oil are explained in detail for the first time. It is observed that three types of operational defects such as bushing heating, fan failure and breather failure can lead to the increase of methanol concentration in oil. Furthermore, the associations of annual load factor and hotspot temperature with methanol are investigated. Besides, various hotspot temperatures of nine transformers are calculated by the temperature rise guide. The results show that the methanol concentration in oil at the bottom of the winding can indicate the hotspot temperature of the winding. To verify this view, the correlation between methanol concentration and hotspot temperature is also proved by the implementation of accelerated non-uniform thermal aging experiments. In addition, the methanol concentration is found to be unevenly distributed along the axial height of the winding. This work not only provides a novel direction for the maintenance of transformer components, but also gives a further support for the distribution mechanism of methanol in insulating oil.