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Methane (CH4) is one of the key greenhouse gases (GHGs) in the atmosphere with current concentration of 1859 ppb in 2017 due to climate change and anthropogenic activities. Rivers are of increasing concern due to sources of atmospheric CH4. However, knowledge and data limitations exist for field studies of subtropical agricultural river catchments, particularly in southern China. The headspace balance method and the diffusion model method were employed to assess spatiotemporal variations of CH4 diffusive fluxes from April 2015 to January 2016 in four order reaches (S1, S2, S3, and S4) of the Tuojia River, Hunan, China. Results indicated that both the dissolved concentrations and diffusive fluxes of CH4 showed obvious spatiotemporal variations. The observed mean concentration and diffusive flux of CH4 were 0.40 &plusmn, 0.02 &mu, mol L&minus, 1 and 41.19 &plusmn, 2.50 &micro, g m&minus, 2 h&minus, 1, respectively, showing the river to be a strong source of atmospheric CH4. The CH4 diffusive fluxes during the rice-growing seasons were significantly greater than the winter fallow season (an increase of 80.26%). The spatial distribution of CH4 diffusive fluxes increased gradually from (17.58 &plusmn, 1.42) to (55.56 &plusmn, 4.32) &micro, 1 due to the organic and nutrient loading into the river waterbodies, with the maximum value at location S2 and the minimum value at location S1. Correlation analysis showed that the CH4 diffusive fluxes exhibited a positive relationship with the dissolved organic carbon (DOC), salinity, and water temperature (WT), while a negative correlation occurred between CH4 diffusive fluxes and the dissolved oxygen (DO) concentration, as well as the pH value. Our findings highlighted that a good understanding of exogenous nutrient loading in agricultural catchments will clarify the influence of human activities on river water quality and then constrain the global CH4 budget.