Charcoal rot of soybean [Glycine max (L.) Merr.], caused by Macrophomina phaseolina (Tassi) Goid causes yield loss in soybean production worldwide. Development of resistant varieties has been limited, in part, due to a lack of consistent and reliable M. phaseolina assessment methodology. Growth stage at assessment is an important consideration. Four soybean lines (DT97-4290, DS97-84-1, Magellan, and A5979) were evaluated for their disease response at four stages of development (R6, R7, R8, and R8 + 1 wk) in order to determine the optimal developmental stage for disease assessment. The soybean lines were sown on 6 May 2005 at Stoneville, MS and artificially inoculated with M. phaseolina at planting. The experimental design was a 4 x 4 factorial in a completely randomized design, where each line-growth stage combination had a minimum of 16 replications. Single plant assessments were made by estimating disease severity present in root and stem tissues using a scale of 1 (resistant) to 5 (susceptible). Over all lines, ratings taken at R8 and R8 + 1 wk had higher disease severities than those taken at R6 and R7. Ratings at R7 had higher severity than those taken at R6. Over all growth stages, DS97-84-1 was most susceptible and DT97-4290 was most resistant. There was no difference among lines at R6 but significant differences among lines occurred at the other three stages. Analysis indicated that stages R7 and R8 were best for distinguishing genotypic differences. Selecting which of the two stages to use may depend on the specific objectives of the research. For example, differentiating among levels of resistance might be most effective at R8, when DT97-4290 was more resistant than A5979, Magellan, and DS97-84-1. And differentiating among levels of susceptibility might be most effective at R7, when DS97-84-1 was more susceptible than Magellan, A5979, and DT97-4290.