Dalmatian pyrethrum (Tanacetum cinerariifolium (Trevir.) Sch. Bip) occurs naturally in the coastal and peri‐coastal regions of the Balkan Peninsula. Due to the production of pyrethrin (insecticide and repellent) in the flower heads, the species has been intensively cultivated in certain regions of the world (Japan, Australia, African countries) over the last 50‐100 years. Using twelve newly developed microsatellite markers, we investigated the differences in genetic diversity and structure of five natural populations from Croatia and one cultivated population from Tasmania. In the populations studied, a total of 102 alleles were detected with an average of 8.5 alleles per locus, indicating high genetic diversity. In the natural populations, a total of 32 private alleles were observed, with the highest number observed in the northernmost (Cres) and southernmost (Konavle) population (10). In the cultivated population from Tasmania, 4 private alleles were found. Both observed and expected heterozygosity in the Tasmanian population was the lowest in the sample (0.442 and 0.455, respectively), closely followed by the Biokovo population (0.466 and 0.478, respectively) which is geographically isolated from the rest of the populations in the native range. The results of the Bayesian analysis of population structure show a clear separation of the Tasmanian and Biokovo populations (each dominated by a separate gene pool) from the natural populations of Croatia at K=3. The neighbour‐joining tree based on Cavalli‐Sforza chord distances showed that the Tasmanian population is closest to the Mali Lošinj population, possibly indicating the original source of material for the large‐scale cultivation of Dalmatian pyrethrum in Tasmania. The results of this research could potentially contribute to an optimal selection of Croatian Dalmatian pyrethrum germplasm to improve the genetic diversity of the impoverished cultivated material in Tasmania, which could facilitate crop adaptation to climate change. This research was funded by the project KK.01.1.1.01.0005 Biodiversity and Molecular Plant Breeding, at the Centre of Excellence for Biodiversity and Molecular Plant Breeding (CoE CroPBioDiv), Zagreb, Croatia.