Although more than 400 microsatellite loci are currently available for Vicia faba L. (faba bean), an important food and feed grain crop legume, they have not yet been used for comprehensive molecular characterization of this crop. We report a three-step procedure for rapid and cost-effective delineation and utilization of informative genomic nuclear SSRs for paralleled genotyping in faba bean suitable also for other species: (i) pre-selection of loci generating PCR products of expected lengths which are potentially polymorphic (achieved by PCR amplification in bulked samples); (ii) exclusion of loci burdened with persistent null alleles and multilocus amplification products (based on PCR amplification of pre-selected loci in individual genotypes), and (iii) multiplexing. We demonstrate also that genomic SSRs are promising molecular tools for molecular characterization of faba bean required also for crop improvement. © 2015, Czech Journal of Genetics and Plant Breeding. All right reserved.