In mammalian cells, DNA replication proceeds according to a precise temporal order during the S phase, but how this program is controlled remains poorly understood. We analyzed the replication-dependent bromodeoxyuridine banding of chromosomes in Chinese hamster cells treated with the spindle poison nocodazole. In these cells, nocodazole induces a transient mitotic arrest, followed by DNA re-replication without intervening cell division. Nuclear fragmentation is often observed in tetraploid derivatives, and previous studies suggest that replication timing of chromosomes could be affected when they are segregated into different micronuclei. Here we show that the onset of replication is frequently asynchronous on individual chromosomes during the re-replication process. Moreover, fluorescence in situ hybridization analysis revealed that replication synchrony is equally altered in fragmented and non-fragmented nuclei, indicating that asynchronous onset of replication is not dependent on physical separation of the chromosomes into isolated compartments. We also show that the ordered program of replication is always preserved along individual chromosomes. Our results demonstrate that the onset of replication of individual chromosomes in the same nuclear compartment can be uncoupled from the time of S-phase entry and from the programmed replication of chromosome sub-domains, revealing that multi-level controls contribute to establish replication timing in mammalian cells.