In general, at the level of extended chromatin domains, replication timing is accurately reproduced in subsequent cell generations. Within these domains, different replication origins fire during each cell cycle, but the choice of origins for replication is probabilistic. Drosophila polytene chromosomes (PCh) are widely used as a model to delineate the functional organization of interphase chromosomes. Earlier we showed, that in general the spatio-temporal organization of replication in Drosophila chromosomes is closely related to the division of the genome into two types of domains, the rb-bands (the most compact PCh bands) and the intervals in between them (INTs, corresponding to alternating zones of loose bands and interbands) (Kolesnikova et al., 2018). In cell cultures and in salivary glands cells INTs correspond to early replication initiation zones. Rb-bands are free of replication initiation events and show a replication timing gradient. In order to verify the applicability of this generic model to individual chromosome regions, we visualized the very beginning of S phase in PCh by super-resolution microscopy. To visualize replication, we used EdU incorporation. In order to investigate the chromosomes at the very beginning of S phase, we used a Drosophila line carrying the hsp70-CycE transgene (Duronio, O'Farrell, 1995) allowing us to induce S phases by heat shock. 3D-structured illumination microscopy (3D-SIM) was used for the ultrastructural investigation of DNA replication. At the beginning of the S phase, no replication occurs inside of the rb-bands. We found EdU signals in INTs, but the intensity and distribution of the signal within the zones were varying. In some zones, a bright band-shaped signal perpendicular to the axis of the chromosome occurred. Usually the signals were laid directly in loose bands and puffs, i.e. in PCh structures commonly associated with the transcription of genes localized inside of them. For the distal part of chromosome 2L we found that the regions, labeled by bright band-shaped EdU signals correspond to high and wide peaks of “early origins” in S2 cells (MacAlpine et al., 2010). The other INTs contain relatively evenly distributed EdU signals over their entire regions, i.e. we identified broad replication initiation zones. Our data verify the previously proposed model of the spatio-temporal organization of replication in Drosophila chromosomes (Kolesnikova et al., 2018) and indicate that the multi-stranded nature of PCh provides a unique opportunity to visualize in one chromosome the stochastic initiation of replication in some regions of the chromosomes, as well as the effective initiation of replication in other sites Funding: Russian Foundation for Basic Research #17-00-00284Duronio RJ, O'Farrell PH // Genes Dev. 1995. V. 9. P. 1456-1468. Kolesnikova TD, Goncharov FP, Zhimulev IF // PLoS One. 2018. V. 13. e0195207. MacAlpine HK, Gordân R, Powell SK et al. // Genome Res. 2010. V. 20. P. 201-211.