Background: Macrophomina phaseolina (Tassi) Goid is a necrotrophic soil-inhabiting fungus that causes disease in several economic plants worldwide and has now become a serious threat to soybean cultivation in central India. Methods: The random surveys were done during 2018 and 2019 to determine the present status of charcoal rot (M. phaseolina) in key soybean growing districts of Madhya Pradesh. Sixteen isolates of pathogen were characterized based on morpho-cultural and pathogenic variability by using cut stem inoculation techniques at J.N.K.V.V., Jabalpur. Result: Charcoal rot was prevalent in all surveyed sixteen districts of Madhya Pradesh. Out of six, Kymore plateau and Satpura hills (44.5%), Satpura plateau (42.5%), Central Narmada Valley (42.0%) and Vindhyan plateau agroclimatic zones (35.0%) were identified as favouable zones for charcoal rot. Malwa plateau (16.25%) and Northern hill region (24.5%) had low incidences of charcoal rot. Among varieties, JS 95-60, JS 93-05 and JS 20-29 were highly affected, whereas, JS 20-34, JS 20-98 and JS 20-69 were least affected. Incidence of charcoal rot was comparatively higher in the fields that had Maize-Chickpea (26.32%), Maize-Wheat, Soybean- Pea and Soybean - Chickpea cropping patterns in the previous year and lowest in Rice-Chickpea (12.00%) and Rice - Wheat (12.45%) cropping patterns. The incidence of charcoal rot was partially higher in the fields with light soil (21.3%) than in heavy soil (19.0%). Isolates investigation revealed that isolates from Jabalpur, Hoshangabad, Chhindwara and Sagar were fast-growing variables and highly aggressive in developing necrotic lesions on the cut stem of soybean. This investigation could be instrumental in forewarning sensitive areas of charcoal rot, varietal options and crop rotation to be followed to minimize the incidence of charcoal rot. Moreover, identified variable aggressive isolates could be utilized in genotype resistance screening programs.