DNA replication is a fundamental process that is primarily regulated at the initiation step. In higher eukaryotes, the location and properties of replication origins are not well understood. Existing genome-wide approaches to map origins—such as nascent strand abundance mapping, Okazaki fragment mapping, or chromatin immunoprecipitation-based assays—average the behavior of a population of cells. However, due to cell-to-cell variability in origin usage, single molecule techniques are necessary to investigate the actual behavior of a cell. Here, I investigate the feasibility of using three single molecule, genome-wide technologies to map origins of replication. The Pacific Biosciences Single Molecule Real-Time (SMRT) sequencing technology, the BioNano Genomics Irys optical mapping technology, and the Oxford Nanopore Technologies MinION nanopore sequencing technology are promising approaches that can advance our understanding of DNA replication in higher eukaryotes.