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In the present study, a diverse set of 96 lentil accessions including biofortified variety IPL 220 was used to identify sources of high concentration for Fe and Zn along with low phytic acid concentration and high protein content. Further, Marker Trait Association (MTA) analysis was performed to find molecular markers controlling these nutritional traits. The average Fe, Zn and phytic acid concentration and protein content ranged from 46.5 to 94.8 (mg/kg), 21.5 to 45.3 (mg/kg), 3.7 to 9.3 (mg/kg) and 18.1–28.7 (%), respectively over the locations. This study showed variability for these traits as coefficient of variation was 11.1% for Fe, 18% for Zn, 18.3% for phytic acid and 8.71% for protein content over two locations. The standard error (S.E.) of mean ranged from 0.05 to 21.5 for Fe, 0.15 to 9.71 for Zn, 0.05 to 2.10 for phytic acid and 0.06 to 6.16 for protein content further calculated across the locations. MTA analysis following general linear model resulted in the identification of 31 MTAs for three traits at p < 0.05 explaining 10 to 26% of total phenotypic variation. Association of PBA LC0203 marker with Fe concentration explained the highest proportion of total phenotypic variation (26%) at Kanpur location. However, same marker with different loci also showed association with Fe concentration at Bhopal location explaining 11% of total phenotypic variation. This study also identified association of some genetic markers with more than two nutritional traits that provides opportunity of improving more than two positively associated nutritional traits through marker assisted selection simultaneously. In the present study, EST-SSR markers were used as functional markers for developing high yielding bio-fortified lentils. • Fe and Zn concentration ranged 47 − 95 and 22 - 45 (mg/kg), respectively. • Phytic acid and protein ranged 4 - 9 (mg/kg) and 18-29 (%), respectively. • CV (%) was 11 for Fe, 18 for Zn, 18 for PA and 9 for protein. • PBA LC0203 marker associated with Fe concentration. • Biofortified lentil variety, IPL 220 was high in Fe and Zn but low in phytic acid. [ABSTRACT FROM AUTHOR]