Groundnut is prone to iron (Fe) deficiency, particularly in calcareous soils, which severely affect growth and productivity. The soils of major groundnut producing areas of Pakistan are calcareous in nature, resulting in limited yield. Hydroponics experiments were performed aiming at the selection of groundnut genotypes with better ability to grow in calcareous soils. For that purpose, 20 locally grown genotypes were screened for their tolerance in terms of morpho-physiological parameters against iron (Fe) deficiency through hydroponics experiments. Various morpho-physiological parameters revealed BARI-2000 and 96CG009 as Fe deficiency tolerant genotypes, whereas BARD-699 and ICGS17 as Fe deficiency sensitive genotypes. The Fe-reduction capacity of roots of these genotypes was also estimated at 2, 4, 6 and 8 day interval. The results depicted the highest Fe-reduction capacity at day 4 among all the genotypes, which decreased at day 6 and 8. At day 2, Fe-reduction capacity of 96CG009 was found higher representing its early response to Fe deficiency. Fe-reduction capacity of BARI-2000 was the highest indicating its tolerance to Fe deficiency, whereas BARD-699 was sensitive to Fe deficiency. The genetic differences among groundnut genotypes were analyzed using thirty SSR markers. These markers amicably differentiated all the genotypes resulting in three main clusters. The phylogenetic analyses based on SSR markers data revealed that Fe deficiency tolerant genotypes tended to cluster together. This implicates that molecular markers can be used for selection of groundnut genotypes with better traits.