Gentiana is an important genus of around 360 medicinally important species, majority of which are not well characterized. Despite its importance, very few genomic resources are available for Gentiana L. Till date, the number of informative and robust simple sequence repeat (SSR)-based markers is limited and very few efforts have been made for their development. A set of robust, freely accessible and informative SSR markers for Gentiana is a pre-requisite for any molecular systematic as well as improvement studies in this group of pharmacologically valuable plants. In view of the importance of these plants, Expressed Sequence Tag (EST) sequences of 18 Gentiana species were surveyed for the development of a large set of non-redundant SSR markers. A total of 5808 perfect SSR with an average length of 17 bp and relative abundance of 214 loci/Mb were identified in the analysed 47,487 EST sequences using Krait software. Mapping of the ESTs resulted in gene ontology annotations of 49.14% of the sequences. Based on these perfect SSRs, 2902 primer pairs were designed, and 60 markers were randomly selected and validated on a set of Gentiana kurroo Royle accessions. Among the screened markers, 39 (65%) were found to be cross-species transferable. This is the first report of the largest set of functional, novel genic SSR markers in Gentiana, which will be a valuable resource for future characterization, genotype identification, conservation and genomic studies in the various species of this group of important medicinal plants.