In metazoans, development and cell differentiation are known to affect various aspects of chromosomal organization at developmentally regulated gene loci (e.g., nuclear localization, locus accessibility, chromatin modifications, etc.). Recent evidence also indicates that cell differentiation can have dramatic effects on DNA replication initiation and replication fork progression, at several of these loci. Hence, origin selection and activation are no longer viewed as constitutive events, but as actively regulated processes, the deregulation of which could potentially affect gene expression and genomic stability at particular chromosomal locations. For these reasons, the study of DNA replication across large genomic regions has recently received increasing attention. Here we describe an assay based on the analysis of single DNA molecules that can be used to determine the steady-state distribution of replication forks across large genomic regions. This assay can be used to study how DNA replication initiates, progresses, pauses, and terminates at single-copy loci in mammalian cells.