DNA synthesis must be performed with extreme precision to maintain genomic integrity. In mammalian cells, different genomic regions are replicated at defined times, perhaps to preserve epigenetic information and cell differentiation status. However, the molecular principles that define this S phase program are unknown. By analyzing replication foci within discrete chromosome territories during interphase, we show that foci which are active during consecutive intervals of S phase are maintained as spatially adjacent neighbors throughout the cell cycle. Using extended DNA fibers, we demonstrate that this spatial continuity of replication foci correlates with the genetic continuity of adjacent replicon clusters along chromosomes. Finally, we used bioinformatic tools to compare the structure of DNA foci with DNA domains that are seen to replicate during discrete time intervals of S phase using genome-wide strategies. Data presented show that a major mechanism of S phase progression involves the sequential synthesis of regions of the genome because of their genetic continuity along the chromosomal fiber.