Fusarium wilt, caused by Fusarium oxysporum f. sp. niveum (FON), remains a major threat to watermelon production worldwide. Effective management depends on accurate race identification, as resistance in commercial cultivars is race-specific. However, current bioassay-based race differentiation is unreliable due to genetic variability within isolates. While molecular identification exists for FON Races 1 and 2, confirming Race 3 has required multiple PCR reactions, making diagnostics cumbersome and inefficient. This study developed and optimized a multiplex PCR assay that simultaneously differentiates FON Races 1, 2, and 3 in a single reaction, significantly improving diagnostic speed and accuracy. FON isolates and related Fusarium species from Georgia, Florida, and South Carolina were tested to assess the assay’s sensitivity (0.5 ng/µL detection limit) and specificity. Results confirmed that the multiplex PCR effectively distinguishes FON from non-pathogenic Fusarium species while accurately identifying all three pathogenic races. This is the first successful multiplex PCR assay for FON race differentiation, providing a rapid, reliable tool for plant pathologists and diagnosticians to track the spread of virulent FON races. Given the increasing prevalence of Race 3, which lacks effective fungicidal control, this tool will support early intervention strategies to mitigate outbreaks and inform resistance breeding programs. Keywords: Fusarium oxysporum f. sp. niveum (FON), multiplex PCR, race differentiation, watermelon wilt.
