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DNA nucleobases are the prime targets for chemical modifications by endogenous and exogenous electrophiles. Alkylation of the N7 position of guanine and adenine in DNA triggers base-catalyzed imidazole ring opening and the formation of N5-substituted formamidopyrimidine (N5-R-FAPy) lesions. Me-FAPy-dG adducts induced by exposure to methylating agents and AFB-FAPy-dG lesions formed by aflatoxin B1 have been shown to persist in cells and to contribute to toxicity and mutagenicity. In contrast, the biological outcomes of other N5-substituted FAPy lesions have not been fully elucidated. To enable their structural and biological evaluation, N5-R-FAPy adducts must be site-specifically incorporated into synthetic DNA strands using phosphoramidite building blocks, which can be complicated by their unusual structural complexity. N5-R-FAPy exist as a mixture of rotamers and can undergo isomerization between α, β anomers and furanose-pyranose forms. In this Perspective, we will discuss the main types of N5-R-FAPy adducts and summarize the strategies for their synthesis and structural elucidation. We will also summarize the chemical biology studies conducted with N5-R-FAPy-containing DNA to elucidate their effects on DNA replication and to identify the mechanisms of N5-R-FAPy repair.