Dalmatian pyrethrum (Tanacetum cinerariifolium Trevir./ Sch. / Bip) is an endemic species of the eastern coast of the Adriatic Sea. The plant species is of economic importance due to the content of the natural insecticide pyrethrin, which is composed of six active ingredients pyrethrin I i II, cinerin I i II, and jasmolin I and II. Pyrethrins are used in organic food production due to their low toxicity to humans and warm-blooded animals. This study aimed to determine potential differences in pyrethrin content depending on the Dalmatian pyrethrum flower development stage of the between the two populations (Senj and Biokovo). Matrix Solid Phase Dispersion was used for the extraction of pyrethrins from dried pyrethrum flowers samples, while High Performance Liquid Chromatography was used for the identification and quantification of pyrethrin. The lowest pyrethrin content in both populations was recorded in the first development stage (unopened buds, top of the ray flowers visible). In this stage population from Biokovo contained 0.53 % of pyrethrins in the dry flower head, while in the population of Senj slightly lower values were determined, 0.58 %, respectively. The highest total pyrethrin content was determined in the third development stage (ray flowers developed, disc flowers visible); 0.99 % of the dry flower weight in the population from Biokovo and 1.17 % in the Senj population. The population of Senj in all developmental stages recorded a higher pir I / pir II ratio, and thus higher insecticidal activity compared to the population of Biokovo. The pir I / pir II ratio in the population of Senj continuously increased to the seventh developmental stage, in which its highest value was recorded. The value of the pir I / pir ratio II in the population of Biokovo oscillated through all seven developmental stages, but the highest value was recorded in the first stage.