While some DNA base modifications such as 5-methylcytosine have been known and studied for decades, recent discoveries of a number of other modified bases have stimulated research to understand their origin and function. Chemistry-based methods for their detection and analysis have proven to be important for advancing the field. Here, we feature a selection of methods that have helped advance the field, along with some key advances in the understanding of how the chemistry of modified bases affects biological functions. We also discuss fundamental questions in the field that remain unanswered.