Fusarium wilt, caused by Fusarium oxysporum f. sp. mungcola (Fom), is an increasingly serious disease of mungbean (Vigna radiata (L.) R.Wilczek) in China. Pathogenic variability has been observed among Fom isolates; however, there are no reports describing Fom races or pathotypes. Thus, this study was conducted with the aim of developing a set of pathotype differentials to reveal Fom pathotype diversity by assessing virulence variability of Fom isolates. First, 105 mungbean cultivars were screened against a standard virulent Fom isolate (F08). Eleven of the 105 cultivars were selected as candidate differentials of Fom according to resistance phenotype and genetic background. Second, the resistance of the 11 candidate differential cultivars was tested against 30 Fom isolates from different geographical origins in China. Highly significant differences were observed among isolate × cultivar interaction patterns, indicating that pathotype differentiation exists in Fom isolates. Based on the different reaction patterns combining with genetic background, seven of the 11 cultivars were selected to constitute a set of differential hosts of Fom pathotype, used to distinguish pathotypes of 84 Fom isolates from different geographical regions by evaluating the virulence reaction pattern. The results showed that the 84 Fom isolates were defined as 12 pathotypes. Finally, we tried to confirm whether the 12 Fom pathotypes could be distinguished by a PCR-based diagnostic method based on the two genes (SIX6 and SIX11) reported to be associated with Fom pathogenicity. However, the Fom pathotype could not be distinguished by variation of the PCR products or their resulting sequences of the two genes. This is the first study to develop a set of Fom pathotype differential hosts and identify 12 Fom pathotypes, which provides important information for resistance breeding and disease control.