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Macrophomina phaseolina (Tassi) Goid was isolated from sick field of sunflower (Helianthus annuus L.) grown from Sargodha, Pakistan. Round to oblong shape sclerotia were recorded in morphological characterization. The isolate MIQ was highly virulent in pathogenicity test. Nucleotide evidence of internal transcribed spacer (ITS) regions (ITS1 and ITS2) from MIQ was submitted under the GenBank accession no. MH277017. In evolutionary tree, nucleotide evidence was compared with the charcoal rot of sunflower, mash, potato, sesame, black gram and cotton reported from Pakistan, China and Korea. MIQ shared the clusters with sunflower as it exhibited 99% genetic homology with charcoal rot of sunflower. Seventy nine germplasms of sunflower (61 from NARC and 18 from CA Sargodha) were screened during two consecutive years 2016-17 and 2017-18, against MIQ to figure out the best resistance sources under the field conditions. During the first year, the screening results revealed 0, 2, 5, 9, 8, 35 and 20 germplasms/lines found to be immune, highly resistant, resistant, moderately resistant, moderately susceptible, susceptible and highly susceptible respectively. During the second year, this response was changed to 0, 2, 3, 5, 6, 41 and 22 germplasms/lines respectively. Out of 61 lines received from NARC, none of the lines showed immune, highly resistant while 17576 and 17586 were found resistance. 5, 4, 32 and 18 were moderately resistance, moderately susceptible, susceptible and highly susceptible respectively. Out of 18 lines, B8555 and B2728 found to be highly resistant, B-208, B-302 and Rafinjan Black Iran exhibited resistant response and B-224, HA-259, HA-65 and Pervenate responded as moderately resistant. During the second year, the change in resistance to the susceptible response of NARC (17576, 17586, 17551, 17565, 17555 and 17546) and COA (Pervenat) lines/ germplasms were recorded. Thus the germplasms of COA, Sargodha consisted of a greater number of resistant lines as compared to that of NARC, Islamabad and can be used in sunflower breeding program against destructive charcoal rot disease of sunflower.