Iron (Fe) deficiency is one of the common problems in strawberry (Fragaria x ananassa Duch.) growing regions. In vitro screening enables researchers to evaluate genotype performance under several concentrations of Fe in a relatively fast and economical way. Therefore, we evaluated the responses of several genotypes belonging to octoploid Fragaria chiloensis (L.) Mill. and Fragaria virginiana Mill. (the progenitors of cultivated strawberry) against Fe treatments under in vitro conditions. In the first experiment, HM 1, Pigeon Point, CFRA1267 (F. chiloensis subsp. pacifica), and CA1541 (F. chiloensis subsp. chiloensis) were tested with various Fe concentrations (0%, 10%, 15%, 25%, 50%, 65%, 80%, and 100%, where 100% = 27.8 mg L-1 FeSO4.7H(2)O). Different Fe concentration treatments resulted in differences in plant height, fresh and dry weight, as well as macro- and microelement concentrations among the tested genotypes. In the second experiment, 16 octoploid Fragaria genotypes were tested with 4 Fe concentration treatments (0%, 10%, 50%, and 100%, where 100% = 27.8 mg L-1 FeSO4.7H(2)O). Iron concentration and genotype interactions were significant for all studied characteristics except sulfur (S) and manganese (Mn) concentrations, and the 16 genotypes responded differentially to varying Fe treatments. The results of these experiments indicated that F. chiloensis and F. virginiana genotypes exhibited considerable variation under different Fe treatments in vitro, and the more resistant genotypes could be utilized to develop new strawberry cultivars with tolerance to low Fe concentrations.