Epitranscriptomic RNA modifications can regulate biological processes, but there remains a major gap in our ability to identify and measure individual modifications at nucleotide resolution. Here we present Mal-Seq, a chemical method for sequencing 5-formylcytosine (fC) modifications on RNA based on the selective and efficient malononitrile-mediated labeling of fC residues to generate adducts that are read as C-to-T mutations upon reverse transcription and polymerase chain reaction amplification. We apply Mal-Seq to characterize the prevalence of fC at the wobble position of mt-tRNA(Met) in different organisms and tissue types and find that high-level fC modification is present in mammals but lacking in lower eukaryotes. Our work sheds light on mitochondrial tRNA modifications throughout eukaryotic evolution and provides a general platform for characterizing the fC epitranscriptome.