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Macrophomina phaseolina is the most devastating and emerging threat to groundnut production in India. An increase in average temperature and inconsistent rainfalls resulting from changing climatic conditions are strongly believed to aggravate the disease and cause severe yield losses. The present study aims to conduct a holistic survey to assess the prevalence and incidence of dry root rot of groundnut in major groundnut growing regions of Southern India, viz., Andhra Pradesh, Telangana, Karnataka, and Tamil Nadu. Furthermore, the pathogenic variability was determined using different assays such as morphological, cultural, pathogenic, and molecular assays. Results indicate that disease incidence in surveyed locations ranged from 8.06 to 20.61%. Both temperature and rainfall played a major role in increasing the disease incidence. The pathogenic variability of M. phaseolina isolates differed significantly, based on the percent disease incidence induced on cultivars of JL-24 groundnut and K-6 groundnut. Morphological variations in terms of growth pattern, culture color, sclerotia number, and sclerotia size were observed. The molecular characterization of M. phaseolina isolates done by ITS rDNA region using ITS1 and ITS4 primers yielded approximately 600 bp PCR amplicons, sequenced and deposited in GenBank (NCBI). Molecular variability analysis using SSR primers indicated the genetic variation among the isolates collected from different states. The present investigation revealed significant variations in pathogenic variability among isolates of M. phaseolina and these may be considered important in disease management and the development of resistant cultivars against groundnut dry root rot disease. [ABSTRACT FROM AUTHOR]