Technical Abstract: Mold diseases, caused by fungal complexes including Alternaria, Cochliobolus and Fusarium species, limit sorghum grain production. Media were tested by plating Fusarium thapsinum, Alternaria sp. and Curvularia lunata, individually and competitively. Dichloran chloramphenicol rose bengal (DRBC) and modified V8®-juice (ModV8) agars, found to be useful, were compared to commonly-used agar media, dichloran chloramphenicol peptone (DCPA) and pentachloronitrobenzene (PCNB). Radial growth, starting with mycelia, or single-conidia and hyphal tips, demonstrated an effect of media. For isolation of grain fungi, DRBC and ModV8 were similar or superior to DCPA and PCNB. When seedlings were inoculated with conidia of C. lunata, Alternaria sp., F. thapsinum, or mixtures, percent root infection ranged from 28% to 77%. For mixed inoculations, shoot weights, lesion lengths and percent root infections were similar to F. thapsinum inoculations; most colonies recovered from roots were F. thapsinum. For Alternaria grain isolates, five morphological types, including Alternaria alternata, were distinguished by colony morphologies and conidial dimensions. Sequence analysis using a portion of the endo-polygalacturonase gene was able to further distinguish isolates. Cochliobolus isolates were identified morphologically as C. lunata, Curvularia sorghina and Bipolaris sorghicola. Multiple molecular genotypes were apparent from rRNA internal transcribed spacer region sequences from Cochliobolus grain isolates.