AbstractThe University of Minnesota (UMN) Pyrethrum Breeding Program has been developing Chrysanthemum cinerariifolium with increased levels of Pyrethrin I/Pyrethrin II ratio (%Py) for over a decade. Pyrethrum is an economically important “green pesticide,” producing %Py in glandular trichomes, with the highest concentration in the flowers. The compounds are useful as insecticides for mosquito, deer tick, bed bug, and insects affecting commercial plant production. Selection for early seed germination and flowering in first‐year pyrethrum seedlings successfully selected annualized perennials. The objectives of this research were to determine genetic variation within and among populations of C. cinerariifolium and Chrysanthemum coccineum from commercial seed lots from Africa, Australia, China, South America, the Netherlands, and the United States, as well as a wild population collected in Croatia and resultant F1 hybrids from the UMN Breeding Program. A total of n = 404 genotypes from 58 populations were tested using DArTseqLD (Diversity Arrays Technology) genotype by sequencing to obtain single nucleotide polymorphism (SNP) markers for 10 different groups. SNP analysis of all taxa resulted in 1786 polymorphic DArTseqLD SNP markers with C. coccineum being genetically distinct from all C. cinerariifolium in the principal component analyses. The global germplasm showed low levels of genetic diversity, demonstrating a very narrow germplasm base and confirmed sharing of genetic stock among private and public sector breeding programs and production firms. We found less genetic variation in the UMN germplasm than expected, although levels of heterozygosity increased in more recent pedigrees. However, several divergent outliers were found as potential sources to enhance genetic variation.