N7-methylguanosine (m7G) is a positively charged, essential modification at the 5′ cap of eukaryotic mRNA, regulating mRNA export, translation, and splicing. m7G also occurs internally within tRNA and rRNA, but its existence and distribution within eukaryotic mRNA remain to be investigated. Here, we show the presence of internal m7G sites within mammalian mRNA. We then performed transcriptome-wide profiling of internal m7G methylome using m7G-MeRIP sequencing (MeRIP-seq). To map this modification at base resolution, we developed a chemical-assisted sequencing approach that selectively converts internal m7G sites into abasic sites, inducing misincorporation at these sites during reverse transcription. This base-resolution m7G-seq enabled transcriptome-wide mapping of m7G in human tRNA and mRNA, revealing distribution features of the internal m7G methylome in human cells. We also identified METTL1 as a methyltransferase that installs a subset of m7G within mRNA and showed that internal m7G methylation could affect mRNA translation. Display omitted •Presence of internal m7G in mammalian mRNA•Chemical-assisted m7G-seq maps m7G methylome at single-base resolution•Highly modified internal m7G sites were identified in human mRNA•METTL1 acts as a methyltransferase for a subset of internal m7G sites in mRNA Zhang et al. discovered the presence of internal N7-methylguanosine (m7G) within mammalian mRNA. Both antibody-based and chemical-assisted methods were developed for transcriptome-wide mapping of internal m7G, with the latter reaching single-base resolution. METTL1/WDR4 was identified as a writer complex that installs a subset of m7G on mRNA, which affects translation.