**Background:** Iron (Fe) deficiency affects approximately 2 billion people globally and is a leading cause of anemia. Chickpea (Cicer arietinum L.) is a staple legume in many developing countries and a potential vehicle for Fe biofortification. However, the mechanisms of Fe uptake, translocation, and accumulation into seeds are not well understood in chickpea. This study aimed to evaluate Fe accumulation in organs at different growth stages and assess expression of Fe-metabolism genes across diverse chickpea genotypes.
**Methods:** Six chickpea genotypes (three cultivated: CDC-551-1, CDC Verano, FLIP97-677C; three wild: Kalka 064, Sarik 067, Cermi 075) were grown in a hydroponic system under controlled conditions (16 h/22°C day, 8 h/15°C night; PAR 220 µmol m⁻² s⁻¹). Plants were subjected to Fe-added (5 μM Fe(III)-EDDHA) or Fe-zero (0 μM Fe(III)-EDDHA) conditions at vegetative stages V3 and V3; reproductive stages R2, R5, R6, and RH were grown only under Fe-added. Fe concentration was measured in roots, stems, leaves, and seeds using ICP-atomic emission spectrometry. Gene expression of FRO2, IRT1, NRAMP3, V1T1, YSL1, FER3, WEE1, and GCN2 was analyzed by qPCR in root and leaf tissues at V3, V10, R2, and R5 stages. Statistical analysis used PROC GLM in SAS 9.4.
**Key Results:** Under Fe-zero, Fe concentration in roots decreased from V3 to V10 in all genotypes, with a higher reduction in cultivated (33%–62%) vs. wild species (6%–50%). Under Fe-added, root Fe concentration increased from V3 to V10. Across all growth stages, roots had the highest Fe concentration and stems the lowest. Seed Fe concentration decreased from R5 to RH, but Fe amount increased, with Sarik 067 showing the highest seed Fe concentration (76 µg g⁻¹) and Fe amount (1.7 g) at RH, while Kalka 064 had the lowest (38 µg g⁻¹). Wild species generally accumulated more Fe in seeds than cultivated ones. Root and shoot dry weight were significantly higher (p < 0.05) under Fe-added vs. Fe-zero at both V3 and V10. Gene expression analysis showed FRO2 and IRT1 were more expressed in roots under Fe-added; at V3, CDC 551-1 had 14-, 12-, and 10-fold higher FRO2 expression than wild genotypes Kalka 064, Sarik 067, and Cermi 075, respectively. Transporter genes NRAMP3, V1T1, and YSL1 were predominantly expressed in leaves. Under Fe-zero, IRT1 expression was higher in most genotypes. FER3 showed higher leaf expression under Fe-zero at V3, except in CDC-551-1. WEE1 and GCN2 were upregulated under Fe-zero at V3. Correlation analysis revealed strong positive correlations (p < 0.01) between Fe concentration and FRO2 (r = 0.95 at V3 roots), IRT1 (r = 0.99 at V3 and V10 roots), NRAMP3 (r = 0.95 at V10 roots), and FER3 (r = 0.88 at V10 roots) under Fe-added.
**Clinical Implications:** This study provides foundational knowledge for breeding Fe-biofortified chickpea varieties. The identification of key genes (FRO2, IRT1, NRAMP3, V1T1, YSL1, FER3, GCN2, WEE1) and their expression patterns across tissues and growth stages offers molecular targets for selection or genetic modification. The finding that wild species (e.g., Sarik 067) accumulate higher seed Fe than cultivated genotypes suggests wild germplasm may be a valuable resource for biofortification. Increasing Fe concentration in chickpea seeds could help alleviate Fe deficiency in populations that rely on chickpea as a dietary staple.