Methane (CH₄) is an important greenhouse gas contributing to global climate change. The Three Gorges Reservoir (TGR) is the largest reservoir in China and has been receiving concerns for its potential CH₄ emissions during the past decades. However, the current research in the TGR mainly focuses on the observation of water-air interface fluxes, the knowledge of relationship between bottom sediments in the reservoir and CH₄ concentrations variation of the bottom water column is still lacking. To explore CH₄ concentrations variation and main influencing factors of the bottom water column in the middle section of TGR, this study collects overlying water and sediment of the bottom respectively at Nanling Town of Fuling, Shibaozhai of Zhongxian and Xiaozhou Town of Wanzhou in the middle section of the TGR during August 2017 and November 2018, and analyzing the data combined with reservoir operation characteristics. The results indicate that CH₄ concentrations in the overlying water of the bottom ranges from 0.02 μmol/L to 0.91 μmol/L, carbon dioxide (CO₂) ranges from 0.006 mmol/L to 0.105 mmol/L, the organic carbon (OC) content in the sediment ranges from 7 g/kg to 90 g/kg, and total nitrogen ranges from 0.27 g/kg to 45.6 g/kg. During the low water level period of the TGR (May to September), a large amount of allochthonous OC input from upstream and terrestrial sources is a sufficient condition for CH₄ accumulation of the bottom in the middle section of the TGR. During the high water level period (October to April of the following year), water level and runoff variations have no obvious effect on CH₄ of the bottom in the middle section of the TGR, the relative proportion of autochthonous organic matter of the bottom in the reservoir is increased, and temperature is the main aquatic environmental factor that affected the CH₄ distribution.