Marker genes are required for the genetic manipulation of living organisms. For selecting transformants after the process of transformation, two types of marker genes are employed: one for the positive selection of the transformants carrying the positive selection marker gene and another for the negative selection of the transformants lacking the negative selection marker gene. Here we have summarized the current advancements in genome engineering of Fusarium species. We have focused on introducing arbitrary mutations by applying both positive and negative selection approaches that result in the exclusion of the marker genes from the target genome. We have discussed the advantages and necessity of using this technique in studies on the regulation of biosynthetic gene expression in mycotoxin production.