Fusarium oxysporum f. sp. niveum (FON), the causal agent of Fusarium wilt of watermelon, is a devastating pathogen that is responsible for significant losses of watermelon in many states in the US and in the world. Differentiation of this pathogen from other formae speciales of F. oxysporum based on morphological characteristics is difficult and time-consuming. A few molecular methods have been developed to specifically detect FON, primarily based on conventional PCR. Loop-mediated isothermal amplification (LAMP) provides a cheaper and quicker molecular detection tool compared to traditional PCR, which can also be used in the field for rapid on-site detection. In this study, we developed a highly sensitive LAMP assay to detect F. oxysporum pathogens of watermelon based on the SIX6 avirulence protein gene that can detect a quantity as low as 0.1 pg/?l of genomic DNA. Findings from this study are helpful for designing sound strategies to detect and manage this disease efficiently.