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Ginger (Zingiber officinale Rosc.) is an economically important and valuable spice crop around the world. It is used as food, spice, condiment, and medicine. A considerable extent of genetic diversity in ginger occurs in the Western Ghats and North-Eastern India. However, genetic diversity studies at the molecular level in ginger is limited due to limited availability of genetic and genomic information. In the present study, for the first time, we have identified and validated expressed sequence tag (EST)-simple sequence repeat (SSR) markers from ginger. We obtained 16,790 EST-SSR loci from 78987 unigenes, and 4597 SSR loci in the predicted 76929 coding sequences from RNA-Seq assembled contigs of ginger through Illumina paired-end sequencing. Gene ontology results indicate that the unigenes with SSR loci participate in various biological processes such as metabolism, growth, and development in ginger. One hundred and twenty-five primer pairs were designed from unigenes and coding sequences. These primers were tested for PCR optimization, characterization, and amplification and identified 12 novel EST-SSR markers. Twelve flanking polymorphic EST-SSR primers were validated using 48 ginger genotypes representing North-Eastern India and different eco-geographical adaptations by PCR amplification and allele sizing through capillary electrophoresis. Twelve EST-SSR primers generated a total of 111 alleles with an average of 9.25 alleles per locus and allele sizes ranging between 115-189bp. This study implies that the SSR markers designed from transcriptome sequences provides ample EST-SSR resources, which are helpful for genetic diversity analysis of Zingiberaceae species and molecular verification of ginger genotypes.