Macrophomina phaseolina is a soil-borne pathogen that causes charcoal rot. Charcoal rot is an emerging and significant disease in Australia and in other strawberry producing countries, and associated with the phase-out of methyl bromide. M. phaseolina growth is favoured by temperatures above 300C, it has a wide host range and current soil fumigants have not been effective in controlling the pathogen. Breeding for resistant cultivars against M. phaseolina is therefore an important focus of the national strawberry breeding program in Australia. Twelve cultivars, consisting of current and historical Australian and international genotypes were screened in a glasshouse trial by drenching the potting media with M. phaseolina microsclerotia inoculum. The cultivars tested varied in their response and were placed in four groups: very high resistance (Earlibrite, Earlisweet, Kabarla and Phenomenal); high resistance (Sweet Charlie, Suncoast Delight and Red Rhapsody); low resistance (Rubygem, Florida Festival and Florida Radiance); and very low resistance (Albion and Camarosa). In a subsequent experiment, a total of thirty isolates of M. phaseolina from strawberry, sorghum, mung bean, peanut, chickpea and watermelon were inoculated on strawberry cv. Albion. M. phaseolina isolates collected from strawberry plants and from sorghum in fields used for strawberry production were more aggressive towards strawberry. The findings of this study will be incorporated into the national strawberry breeding program in Australia to develop cultivars with resistance to charcoal rot.