In order to determine the timing and mechanisms of the spontaneous diploidisation throughout microspore-derived embryogenesis in barley, we have estimated the ploidy level of individual nuclei within young pro-embryos, from the first androgenetic division up to multinuclear structures still surrounded by the exine. Our methodological approach was based on the measure of the intensity of fluorescence after 4,6-Diamidino-2-phenylindole dihydrochloride staining, nuclear size and number of nucleoli in the confocal microscope. This method avoids the overlapping of the fluorescence signal in multinuclear pro-embryos, which cannot be studied using cytophotometer methods based on other types of fluorescence microscopes. The identification of haploid and diploid nuclei enabled us to determine the timing of diploidisation at early stages throughout androgenetic development. We found that diploidisation is an ongoing process that can start after the first embryogenic division and continues in multinuclear pro-embryos. Reconstruction of 3D-images of entire pro-embryos and the observation of cross and longitudinal sections across stacks of optical sections, together with correlative light and electron microscopy, provided evidences of nuclear fusion as the main mechanism of diploidisation.