Dalmatian pyrethrum is an autochthonous plant species of the Republic of Croatia. It is widespread along the Adriatic coastal region and islands. The importance of this plant species lies in the content of secondary metabolite pyrethrin, which is why it is recognized and cultivated worldwide. Pyrethrin acts as a natural insecticide on a large number of insects and is mostly localized in the flower heads. The aim of this paper was to optimize the maceration process for the extraction of pyrethrin from Dalmatian pyrethrum and to investigate the impact of different extraction methods (maceration, ultrasonic extraction, and extraction by matrix solid phase dispersion) on the concentration of isolated total pyrethrins, in three natural populations of Dalmatian pyrethrum (Krk, Kozjak and Senj). The optimal maceration conditions for the highest pyrethrin yield were found to be maceration of 0.25 g of plant material in 5 ml of acetone at a speed of 400 rpm for 3 h. Depending on the analyzed population, the pyrethrin content obtained by maceration ranged from 0.29 – 0.62%, %, in the extraction employing ultrasonic technique pyrethrin content ranged from 0.46 – 0.49%, while with the matrix solid phase dispersion method the obtained pyrethrin content ranged from 0.45 – 0.59%. By the application of different methods, the pyrethrin content varied, however, the differences were not significant.