mRNAs are regulated by nucleotide modifications that influence their cellular fate. Two of the most abundant modified nucleotides are N⁶-methyladenosine (m⁶A), found within mRNAs, and N⁶,2′-O-dimethyladenosine (m⁶Am), which is found at the first transcribed nucleotide. Distinguishing these modifications in mapping studies has been difficult. Here, we identify and biochemically characterize PCIF1, the methyltransferase that generates m⁶Am. We find that PCIF1 binds and is dependent on the m⁷G cap. By depleting PCIF1, we generated transcriptome-wide maps that distinguish m⁶Am and m⁶A. We find that m⁶A and m⁶Am misannotations arise from mRNA isoforms with alternative transcription start sites (TSSs). These isoforms contain m⁶Am that maps to "internal" sites, increasing the likelihood of misannotation. We find that depleting PCIF1 does not substantially affect mRNA translation but is associated with reduced stability of a subset of m⁶Am-annotated mRNAs. The discovery of PCIF1 and our accurate mapping technique will facilitate future studies to characterize m⁶Am's function.