Charcoal rot disease caused byMacrophomina phaseolina(Tassi) Goid is one of the most devastating diseases in soybean in India. During 2018, 226 diverse soybean genotypes were evaluated for genetic resistance under hot-spot conditions. Out of them, a subset of 151 genotypes were selected based on Percent Disease Incidence (PDI) and better agronomic performance. Out of these 151 genotypes evaluated during 2019, 43 genotypes were selected based on PDI and superior agronomic performance for further field evaluation and molecular characterization. During 2020 and 2021, these forty-three genotypes, were evaluated for PDI, Area Under Disease Progress Curve (AUDPC), and grain yield. In 2020, genotype JS 20-20 showed least PDI (0.42) and AUDPC (9.37).Highest grain yield was recorded by the genotype JS 21-05 (515.00 g). In 2021, genotype JS 20-20 exhibited least PDI (0.00) and AUDPC (0.00).Highest grain yield was recorded in JS 20-98 (631.66 g). Across both years, JS 20-20 had the least PDI (0.21) and AUDPC (4.68), while grain yield was highest in JS 20-98 (571.67 g). Through MGIDI (multi-trait genotype-ideotype distance) analysis, JS 21-05 (G19), JS 22-01 (G43), JS 20-98 (G28) and JS 20-20 (G21) were identified as the ideotypes with respect to the traits that were evaluated. Two unique alleles, Satt588 (100 bp) on linkage group K (Chromosome no 9) and Sat_218 (200 bp) on linkage group H (Chromosome no 12), were specific for thetwo resistant genotypes JS 21-71and DS 1318, respectively. Through cluster analysis, it was observed that the genotypes bred at Jabalpur were more genetically related.