Dalmatian pyrethrum (Tanacetum cinerariifolium /Trevir./ Sch. Bip.) is an endemic species of the eastern Adriatic from the family Asteraceae. The plant produces pyrethrin, a secondary metabolite that has shown to be an effective natural insecticide with little or no adverse effects on humans. To assess biochemical and genetic diversity, samples were collected from 10 natural populations of Dalmatian pyrethrum along the Adriatic coast and islands representing the species' distribution range. A matrix solid phase dispersion extraction (MSPD) method was used for the extraction of pyrethrin compounds from the dried flower heads of Dalmatian pyrethrum. High performance liquid chromatography with diode array detector was carried out for the determination and quantification of the six pyrethrin compounds and total pyrethrin content. The total pyrethrin content in the samples ranged from 0.10% to 1.35% of the dry flower weight and the pyrethrin I/pyrethrin II ratio ranged from 0.21 to 5.88. Four chemotypes of different pyrethrin extract quality were determined using multivariate statistical methods. A total of 17 microsatellite markers were developed using Next Generation Sequencing (NGS), of which two were classified as highly polymorphic (PIC > 0.70) and eight as moderately polymorphic (PIC > 0.44). The presence of null alleles was detected at four loci. Genetic diversity analysis of 10 Dalmatian pyrethrum populations was performed using 12 microsatellite markers. Evidence of bottleneck events was identified only in the Biokovo population. Analysis of molecular variance showed that the majority of the genetic diversity can be explained by differences between individuals within populations (87.03%). Bayesian analysis of population structure revealed the existence of two gene pools. Spatial analysis revealed that only a small proportion of genetic differentiation can be attributed to isolation by distance (0.7%), while 40.6% of genetic differentiation can be attributed to isolation by environmental distance. The results of this research will contribute to the development of future breeding programs and hopefully commercial varieties of T. cinerariifolium to revive Dalmatian pyrethrum production in Croatia and the region. Furthermore, these results will help to improve conservation strategies for the species.