The knowledge about the level of genetic diversity and population structure in natural populations of Artemisia annuaL. is a primary step in breeding programs for development of new cultivars with higher artemisinin level and better quality of secondary metabolites composition. We used PCR-based “retrotransposon-microsatellite amplified polymorphisms” (REMAPs) to study insertional polymorphism in A. annuagenome to assess genetic variability and population structure in a collection of 118 accessions collected from north and northwest of Iran. Twenty-five primer combinations of 10 retrotransposon and seven ISSR primers amplified a total of 693 clear and unambiguous fragments in the studied accessions. The average number of bands, polymorphic bands, polymorphism, effective number of alleles, Shannon’s information index and expected heterozygosity were 27.72, 24.76, 88.14%, 1.47, 0.42 and 0.28, respectively. The analysis of molecular variance revealed high genetic variation present within sampled geographical regions. Distance-based cluster analysis assigned the studied accessions into four clusters according to their geographical origin, which were also confirmed by principal coordinate analysis. In model-based Bayesian clustering, the maximum value of Δkwas obtained when the collection of 118 assayed A. annuaaccessions assigned into two subgroups (K= 2). The results showed the high genetic variation in the collection of Iranian sweet wormwood which revealed by REMAP markers indicating the reliability and efficiency of this marker system for analysis of genetic diversity and population structure of A. annua.