Abstract: This invention relates to improved methods and kits for identification of 5-formylcytosine (5fC) to be distinguished from cytosine (C) in a sample nucleotide sequence. Methods comprise reducing a first portion of polynucleotides which comprise the sample nucleotide sequence; treating the reduced first portion and a second portion of polynucleotides with bisulfite; sequencing the polynucleotides in the first and second portions of the population to produce first and second nucleotide sequences respectively and; identifying the residues in the first and second nucleotide sequences which correspond to a cytosine residue in the sample nucleotide sequence. These methods may be useful, for example in the analysis of genomic DNA and/or of RNA.