N⁶,2′-O-dimethyladenosine (m⁶Am), a terminal modification adjacent to the mRNA cap, is a newly discovered reversible RNA modification. Yet, a specific and sensitive tool to directly map transcriptome-wide m⁶Am is lacking. Here, we report m⁶Am-seq, based on selective in vitro demethylation and RNA immunoprecipitation. m⁶Am-seq directly distinguishes m⁶Am and 5′-UTR N⁶-methyladenosine (m⁶A) and enables the identification of m⁶Am at single-base resolution and 5′-UTR m⁶A in the human transcriptome. Using m⁶Am-seq, we also find that m⁶Am and 5′-UTR m⁶A respond dynamically to stimuli, and identify key functional methylation sites that may facilitate cellular stress response. Collectively, m⁶Am-seq reveals the high-confidence m⁶Am and 5′-UTR m⁶A methylome and provides a robust tool for functional studies of the two epitranscriptomic marks.