In addition to the four canonical nucleobases of A, G, T and C, hundreds of chemical modifications have been identified in genome DNA. Among them, 5-formylpyrimidines, including 5-formylcytosine(5fC) and 5-formyluracil(5fU), are naturally occurring pyrimidine-covalent-modifications and are widely distributed in mammalian cells. Emerging evidence indicates that 5fC not only serves as a key intermediate in active DNA demethylation but also carry independent epigenetic significance; while 5fU was always considered to be an oxidative DNA lesion with high genotoxicity. In order to further understand their regulatory functions, it is necessary to develop accurate, sensitive and facile methods for qualitative, quantitative, and localized detection of 5-formylpyrimidines in the whole genome. In this review, we summarized the recent progress in detecting 5-formylpyrimidines from the perspective of selective chemical labeling.