Simple sequence repeats (SSRs) are highly polymorphic and co-dominant markers, providing an important genomic resource for genetic research. Recently, large-scale transcriptome sequencing has become a reliable and efficient approach for the identification and development of new genic-SSR markers and has been successfully conducted in a few important plant species. However, SSR development based on transcriptome sequencing remains limited in radish (Raphanus sativus L.). In the present study, from a total of 73,084 unigenes and 150,455 contigs which were assembled from 71.95 million Illumina sequence reads of a radish taproot library, a collection of 11,928 genic-SSR loci were successfully identified in 11,311 unigene sequences. Trinucleotide repeats were the most abundant repeat units, as in many other plants, with a frequency of 52 %. Furthermore, a total of 5,503 genic-SSR primers were developed, from which 1,052 SSR primers were synthesized, and a subset of 823 (78.23 %) primers could generate stable bands. Moreover, 67 selected informative genic-SSR markers were used to determine the genetic diversity of 32 radish genotypes, in which the polymorphism information content values ranged from 0.49 to 0.89. For effective cultivar identification, a novel strategy called manual cultivar identification diagram was employed. Thirty-two radish accessions were clearly separated by six genic-SSR markers. Additionally, the cross-species/genera transferability of these SSRs was further validated in nine relatives in Brassicaceae. These results suggested that the novel genic-SSR markers, as a basis for future genetic linkage and gene tagging analysis, could be very valuable in facilitating genetic mapping, marker-assisted selection and comparative genome analysis. © 2013 Springer Science+Business Media Dordrecht.