Methane (CH₄), an important greenhouse gas, significantly impacts the local and global climate. Our study focused on the composition and activity of methanotrophs residing in the lakes on the Tibetan Plateau, a hotspot for climate change research. Based on the field survey, the family Methylomonadaceae had a much higher relative abundance in freshwater lakes than in brackish and saline lakes, accounting for ~92% of total aerobic methanotrophs. Using the microcosm sediment incubation with ¹³CH₄ followed by high throughput sequencing and metagenomic analysis, we further demonstrated that the family Methylomonadaceae was actively oxidizing CH₄. Moreover, various methylotrophs, such as the genera Methylotenera and Methylophilus, were detected in the ¹³C-labeled DNAs, which suggested their participation in CH₄-carbon sequential assimilation. The presence of CH₄ metabolism, such as the tetrahydromethanopterin and the ribulose monophosphate pathways, was identified in the metagenome-assembled genomes of the family Methylomonadaceae. Furthermore, they had the potential to adapt to oxygen-deficient conditions and utilize multiple electron acceptors, such as metal oxides (Fe³⁺), nitrate, and nitrite, for survival in the Tibet lakes. Our findings highlighted the predominance of Methylomonadaceae and the associated microbes as active CH₄ consumers, potentially regulating the CH₄ emissions in the Tibet freshwater lakes. These insights contributed to understanding the plateau carbon cycle and emphasized the significance of methanotrophs in mitigating climate change.

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