Chromosomal fragile sites are specific loci that exhibit instability visible as gaps and breaks on the chromosome following inhibition of DNA synthesis and are generally categorized into two main classes: rare fragile sites (RFSs) and common fragile sites (CFSs). Under standard conditions, CFSs are typically stable but are prone to breakage in cells subjected to replication stress. In recent years, their role in the generation of gross chromosome rearrangements has become increasingly evident, and fragile sites have now connected to chromosome instability in cancer cells. The connection between CFSs and cancer thus highlights the importance of the regulation of DNA replication to prevent cancer development. The study of fragile sites in the yeast model organism has provided insight into the mechanisms that lead to breakage and genome instability. Through the process of molecular combing, replication dynamics can be observed at fragile sites to further understand the consequence of replication stress on DNA damage.