Fungal polyketide synthases (PKSs) are members of multi-domain enzyme families. They play an important role in the biosynthesis of secondary metabolites known as fungal polyketides. Various types of PKSs are encoded by PKS gene families in different species. PKS genes are usually organized as gene clusters responsible for the production of enzymes that catalyse critical steps in synthesizing various secondary metabolites. Mycotoxins are secondary metabolites produced by various fungal species. A single species can produce multiple various mycotoxins at the same time. Zearalenone (ZEN), whose biosynthesis is catalysed by two PKSs, occurs predominantly in cereal grains. This mycotoxin affects global public health due to the consumption of ZEN contaminated foodstuffs and feeds in the diets of humans and livestock. ZEN is known as a non-steroidal mycotoxin due to the structural similarity to 17-β estradiol and ability to bind to estrogen receptors. Although several fungal species produce ZEN, the main producers are Fusarium graminearum (teleomorph Gibberalle zeae) and F. culmorum. ZEN biosynthetic gene clusters were identified in F. graminearum genome. The proposed gene cluster consists of 11 genes, including two polyketide synthase genes (PKS4 and PKS13) and additional regulatory genes. In this chapter, the current status of PKS4 and PKS13, which are crucial genes for the ZEN biosynthetic pathway, will be discussed. Improving knowledge about the genes associated with ZEN synthesis will contribute to detection and management strategies of ZEN-producing species.