Fusarium tucumaniae is the only known sexually reproducing species among the seven closely related fusaria that cause soybean sudden death syndrome (SDS) or bean root rot (BRR). In a previous study, laboratory mating of F. tucumaniae yielded recombinant ascospore progeny but required two mating-compatible strains, indicating that it is heterothallic. To assess the reproductive mode of the other SDS and BRR fusaria, and their potential for mating, whole-genome sequences of two SDS and one BRR pathogen were analyzed to characterize their mating type (MAT) loci. This bioinformatic approach identified a MAT1-1idiomorph in F. virguliforme NRRL 22292 and MAT1-2 idiomorphs in F. tucumaniae NRRL 34546 and F. azukicola NRRL 54364. Alignments of the MAT loci were used to design PCR primers within the conserved regions of the flanking genes APN1 and SLA2, which enabled primer walking to obtain nearly complete sequences of the MAT region for six MAT1-1 and five MAT1-2 SDS/BRR fusaria. As expected, sequences of the highly divergent 4.7 kb MAT1-1 and 3.7 kb MAT1-2 idiomorphs were unalignable. However, sequences of the respective idiomorphs and those that flank MAT1-1 and MAT1-2 were highly conserved. In addition to three genes at MAT1-1 (MAT1-1-1, MAT1-1-2, MAT1-1-3) and two at MAT1-2 (MAT1-2-1, MAT1-2-3), the MAT loci of the SDS/BRR fusaria also include a putative gene predicted to encode for a 252 amino acid protein of unknown function. Alignments of the MAT1-1-3 and MAT1-2-1 sequences were used to design a multiplex PCR assay for the MAT loci. This assay was used to screen DNA from 439 SDS/BRR isolates, which revealed that each isolate possessed MAT1-1 or MAT1-2, consistent with heterothallism. Both idiomorphs were represented among isolates of F. azukicola, F. brasiliense, F. phaseoli and F. tucumaniae, whereas isolates of F. virguliforme and F. cuneirostrum were only MAT1-1 and F. crassistipitatum were only MAT1-2. Finally, nucleotide sequence data from the RPB1 and RPB2 genes were used to date the origin of the SDS/BRR group, which was estimated to have occurred about 0.75 Mya (95% HPD interval: 0.27, 1.68) in the mid-Pleistocene, long before the domestication of the common bean or soybean.