Macrophomina phaseolina (Tassi) Goid. is a serious pathogen of charcoal rot disease in the maize plant recently in Punjab, Pakistan. In order to initiate monitoring of this pathogen, 24 isolates of M. phaseolina from four districts of Punjab province of Pakistan were collected to assess the variations in morphology and virulence on the maize plant. Regarding to the geographic origins, significant differences were observed in radial mycelial growth, sclerotial size, sclerotial population per unit area and virulence among the isolates. Thirteen isolates were rated as fast growing (77.10?88.10 mm), seven as medium growing (66.00–77.00mm), and the rest as slow growing (54.00?65.00 mm). Eight isolates produced large sized sclerotia (> 45µm), eleven produced medium sized (40?45 µm), and the remaining five isolates produced small sized sclerotia (