Background Microspore embryogenesis and cytoplasmic male sterility system (CMS) are two approaches widely exploited in Brassica napus breeding for production of homozygous doubled haploid (DH) lines and F1 hybrids respectively. Cytoplasmic male sterility system (CMS) is one of the most important pollination systems for hybrid seed production and utilisation of doubled haploid system to quickly prepare fully homozygous fertility restorer lines for CMS Ogu-INRA is very beneficial. Generally, only a small part of microspore-derived embryos is used for plant regeneration, without any knowledge about their properties. Therefore, the possibility of early detection of desirable genotypes bearing a single dominant nuclear fertility restorer (Rfo) gene, can double the success of selection and reduce the production costs. Results To maximize the efficiency and yield of regenerated microspore-derived embryos (MDEs) with the Rfo gene, a protocol for reliable and early, non-destructive selection of desired MDE genotypes was developed. The total amount of 636 cotyledonary embryos was tested by PCR, out of which 37% (237/636) were shown to bear the Rfo gene (instead of 50% according to the expected 1:1 segregation ratio for a single copy gene) and 218 of these fertility restorer plants were fully grown to flowering stage. New molecular marker has been demonstrated to have 100% of co-segregation with the phenotypic evaluation. Conclusion Technique developed in this study provides early and non-destructive sampling of embryonic tissue and the use of new markers for simple and efficient control of the presence of Rfo gene in all accessions.