The chemical modifications of DNA are of pivotal importance in the epigenetic regulation of cellular processes. Although the function of 5-methylcytosine (5mC) has been extensively investigated, the significance of 5-hydroxymethylcytosine (5hmC) has only recently been acknowledged. Conventional methods for the detection of DNA methylation frequently lack the capacity to distinguish between 5mC and 5hmC, resulting in the combined reporting of both. The growing importance of 5hmC has prompted the development of a multitude of methods for the qualitative and quantitative analysis of 5hmC in recent years, thereby facilitating researchers' understanding of the mechanisms underlying the onset and progression of numerous diseases. This review covers both established and novel methods for the detection of cytosine modifications, including 5mC, 5hmC, 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC), with a particular focus on those that allow for accurate mapping and detection, particularly with third-generation sequencing. The review aims to help researchers choose the most appropriate methods based on their specific research goals and budget.