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Fungi play a unique role in the Earth's biomass. Accurate genetic characterization is an important endeavour in many fields including human health, crop sciences and breeding, and for the development of industrial applications. Transboundary plant fungal diseases affect food crops, cause significant losses to farmers, and threaten food security. Responding to these threats requires improved surveillance and diagnostic systems. Yet, many methods are not suitable for developing countries with varying levels of laboratory infrastructure. Advances in molecular genetics have opened the way for DNA analysis to be incorporated into taxonomy, which has sometimes challenged the historical groupings that were based on morphology and other traits. However, several molecular assays can be employed for the characterization of natural and induced nucleotide variation in fungal pathogens. These help provide a better understanding of gene function and allow a reduction in the time needed to detect emerging and re-emerging new pathotypes.