Background: Bacteria associated with plants can enhance the plants’ growth and resistance against phytopathogens. Today, growers aim to reduce the use of mineral fertilizers and pesticides. Since phytopathogens cause severe yield losses in crop production systems, biological alternatives gain more attention. Plant and also seed endophytes have the potential to influence the plant, especially seed-borne bacteria may express their beneficiary impact at initial plant developmental stages. In the current study, we assessed the endophytic seed microbiome of seven genetically diverse barley accessions by 16S rRNA gene amplicon sequencing and verified the in vitro plant beneficial potential of isolated seed endophytes. Furthermore, we investigated the impact of the barley genotype and its seed microbiome on the rhizosphere microbiome at an early growth stage by 16S rRNA gene amplicon sequencing. Results: The plant genotype displayed a significant impact on the microbiota in both barley seed and rhizosphere. Consequently, the microbial alpha- and beta-diversity of the endophytic seed microbiome was highly influenced by the genotype. Interestingly, no correlation was observed between the endophytic seed microbiome and the single nucleotide polymorphisms of the seven genotypes. Unclassified members of Enterobacteriaceae were by far most dominant. Other abundant genera in the seed microbiome belonged to Curtobacterium, Paenibacillus, Pantoea, Sanguibacter and Saccharibacillus. Endophytes isolated from barley seeds were affiliated to dominant genera of the core seed microbiome, based on their 16S rRNA gene sequence. Most of these endophytic isolates produced in vitro plant beneficial secondary metabolites known to induce plant resistance. Conclusion: Although barley accessions representing high genetic diversity displayed a genotype-dependent endophytic seed microbiome, a core seed microbiome with high relative abundances was identified. Endophytic isolates were affiliated to members of the core seed microbiome and many of them showed plant beneficial properties. We propose therefore that new breeding strategies should consider genotypes with high abundance of beneficial microbes.