Charcoal root rot-like symptoms were observed on Mexican lime (Citrus x aurantiifolia) plants in a nursery located in Hormozgan province, southern Iran. The fungus was identified as Macrophomina phaseolina based on morphological and molecular characteristics. Pathogenicity tests revealed the association of six M. phaseolina isolates with disease. Reisolation from roots of inoculated plants yielded isolates of M. phaseolina with morphological characteristics identical to those of the original isolates used for inoculations, thus fulfilling Koch's postulates. This is the first record of charcoal rot caused by M. phaseolina on citrus in Iran.