Immortelle (Helichrysum italicum (Roth) G. Don; Asteraceae) is a versatile medicinal and aromatic plant (MAP) known for its various biologically active compounds and its high economic importance in different industries. The availability of the H. italicum de novo assembled transcriptome enables the development of expressed sequence tag–simple sequence repeat (EST-SSR) markers, which provide an efficient tool for genetic diversity and molecular marker-assisted selection in MAPs¹. An analysis of transcripts from the de novo transcriptome assembly that contained microsatellite motifs, for which primers have been successfully designed and have a known putative function, revealed a total of 1,999 perfect microsatellites, excluding mononucleotide motifs. Trinucleotide microsatellite motifs had the highest frequency of 70.03% (1,398), followed by dinucleotides (24.26%; 487), tetranucleotides, hexanucleotides, and pentanucleotides. Based on an additional manual examination of the annotated functions, 78 EST-SSRs were selected for an initial screening of eight immortelle samples. The results indicated that 38.5% of the EST-SSRs were successfully amplified using the Schuelke method². Of the 30 successfully amplified EST-SSRs, 23 exhibited polymorphisms, while the remaining 7 EST-SSRs were monomorphic. Within the polymorphic EST-SSRs, 73.9% were located in the coding sequence regions (CDSs), 17.4% in the 3’ untranslated regions (3’ UTRs), and 8.7% in the 5’ untranslated regions (5’UTRs). In the continuing study, the 23 polymorphic EST-SSRs selected after the initial screening were further tested for their potential marker transferability to closely related Helichrysum species (Helichrysum litoreum Guss., Helichrysum arenarium (L.) Moench). Based on their genetic diversity parameters and cross-species transferability, a final set of 12 EST-SSRs was used to characterize the natural populations of immortelle in the Mediterranean region of Corsica (France) and the Istrian peninsula (Croatia). The results of the characterization of new genic microsatellite markers for immortelle and the analysis of natural populations will be presented.