Methane, an important greenhouse gas, has a profound impact on global climate change. Rivers are crucial sites for methane production, and the complexity of river environmental conditions makes it difficult to effectively conduct in-depth research on methane generation by river microorganisms using traditional methods. This article first outlines the basic classification of methanogenic microbes, their distribution and the pathways of methane formation. Then, it explores the environmental factors that affect the methane generation process of microbes. After that, it summarizes the application of molecular biology techniques in the study of river microbial methane production. Finally, it discusses the future research on river methane formation from a mechanistic perspective, providing new ideas and methods for an insight into the exploration on river microbial methane production.