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Early diagnosis of fungal pathotypes is one of the most important aspects for plant breeders. It provides a rapid means for selecting the correct mutant lines to ensure durability of disease resistance and also for proper field management practices. Traditional diagnostic tools for pathogens have been based on targeted cultures, PCRbased approaches, and/or phenotypic evaluation of disease response in specific plant genotypes. These methods detect only known pathogenic agents, can introduce bias, and can fail to recognize novel variants or races due to their narrow scope. While enzymatic mismatch cleavage has been described for many plant and animal species, the validate that technique for haploid microorganisms is also needed. In this work, optimized low cost method for pathotyping Fusarium oxysporum f. sp. cubense (Foc), the causal agent of fusarium wilt of banana (Musa spp.), was established using self-extracted nuclease and agarose gel electrophoresis. Gene-specific primers were designed from the whole fungal genome for use in the enzymatic mismatch cleavage on Foc representatives of the major races of major banana producing countries. Gene-specific primer pairs were used to optimize enzymatic mismatch cleavage and polymorphism discovery in two SNF1 and FOW2 genes. The protocol is rapid, inexpensive and can robustly distinguish pathotypes in Foc strains, without high informatics load of DNA sequencing.