Charcoal rot disease caused by Macrophomina phaseolina (Tassi) Goid is one of the most devastating diseases in soybean in India. During 2018, a total of 226 diverse soybean genotypes have been evaluated for charcoal rot resistance under hot-spot conditions. Out of 226 genotypes, a sub set of 151 genotypes were selected based on disease reaction and better agronomic performance. Out of 151 genotypes evaluated during 2019, 43 genotypes were selected based on disease reaction and superior agronomic performance for their further field evaluation and molecular characterization. Disease evaluation during 2018 and 2019 was through Percent Disease Incidence (PDI). During 2020 and 2021, these forty three genotypes, were evaluated for PDI, Area Under Disease Progress Curve (AUDPC), and grain yield. During 2020, genotype JS 20–20 was found to show least PDI (0.42) and AUDPC (9.37), and highest grain yield was recorded in the genotype JS 21 − 05 (515.00 g). During 2021, genotype JS 20–20 observed to exhibit least PDI (0.00) and AUDPC (0.00), and highest grain yield was recorded in JS 20–98 (631.66 g). Across both the years, JS 20–20 was found to show 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 to be the ideotypes with respect to the traits under consideration. Two unique alleles, Satt588 (100 bp) on linkage group K and Sat_218 (200 bp) on linkage group H, were found to be specific to two resistant genotypes JS 21-71and DS 1318, respectively. Through clustering pattern, it was observed that the genotypes bred at Jabalpur are genetically more closely related.