Fusarium solani f. sp. glycines (FSG; syn. F. virguliforme Akoi, O'Donnell, Homma & Lattanzi) is a soil-borne fungus that infects soybean roots and causes sudden death syndrome (SDS), a widespread and destructive soybean disease. The goal of this study was to develop and use a real-time quantitative polymerase chain reaction (QPCR) assay to compare the accumulation of genomic DNA among 30 FSG isolates in inoculated soybean roots. Isolates differed significantly (P < or = 0.05) in their DNA accumulation on a susceptible soybean cultivar when detected and quantified using a FSG-specific probe/primers set derived from the sequences of the nuclear-encoded, mitochondrial small subunit ribosomal RNA gene. QPCR results that were normalized as the fold change over the sample collection times after inoculation were significantly (P < or = 0.001) correlated with the log(10) transformed colony-forming unit (CFU) values of FSG obtained from plating of inoculated ground roots on FSG semi-selective agar medium. Several isolates were identified that accumulated more FSG DNA and had higher CFU values than the reference isolate FSG1 (Mont-1). Compared to other isolates, FSG5 was the most aggressive root colonizer based on DNA accumulation and CFU values in infested roots. The described QPCR assay should provide more specificity, greater sensitivity, and less variability than alternatives to the culturing-dependent and time-consuming plating assays. Evaluation of isolate relative DNA differences on host plants using the QPCR approach provides useful information for evaluating isolates based on the extent and/or degree of colonization on soybean roots and for selecting isolates for breeding SDS-resistant soybean lines.