The combined pressures of overharvesting for ornamental and medicinal markets, along with pervasive habitat degradation, have precipitated a critical threat to wild populations of the jewel orchid, Ludisia discolor , within the biodiversity hotspots of Vietnam. The development of robust molecular tools is therefore essential to inform effective conservation strategies. This study established a suite of polymorphic Expressed Sequence Tag-derived Simple Sequence Repeat (EST-SSR) markers for L. discolor and applied them to assess genetic diversity and population structure. Transcriptome sequencing on the Illumina HiSeq™ 4000 platform yielded 44,764,702 high-quality reads. De novo assembly generated 26,271 unigenes (N50 = 2,160 bp; Q30 = 96.3%), which were comprehensively annotated against public databases (Nr, Swiss-Prot) and classified into Gene Ontology, KOG, and KEGG pathways. From 9,495 identified EST-SSR loci, 15 highly polymorphic markers were validated. Analysis of Vietnamese populations revealed moderate genetic diversity (Na = 3.17; He = 0.54; PIC = 0.59). Analysis of molecular variance (AMOVA) indicated significant genetic differentiation, with 23% of variation partitioned among populations. Bayesian clustering, Principal Coordinates Analysis (PCoA), and Neighbor-Joining (NJ) tree reconstruction consistently identified two distinct genetic clusters correlated with geography, suggesting restricted gene flow. These transcriptome-derived EST-SSR markers have proven effective for population genetic studies in L. discolor . The genomic resources and findings presented here provide a critical foundation for genetic monitoring and support the integration of molecular data into conservation and restoration programs for this ecologically and culturally significant orchid.