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Genotyping by sequencing (GBS) was used to reveal the inherent genetic variation within the haploid fungi <i>Sarocladium zeae</i> isolated from diverse <i>Zea</i> germplasm, including modern <i>Zea mays</i> and its wild progenitors—the teosintes. In accordance with broad host relationship parameters, GBS analysis revealed significant host lineages of <i>S. zeae</i> genetic diversity, indicating that <i>S. zeae</i> genetic variation may associate with different evolutionary histories of host species or varieties. Based on a recently identified PKS-NRPS gene responsible for pyrrocidine biosynthesis in <i>S. zeae</i> fungi, a novel PCR assay was developed to discriminate pyrrocidine-producing <i>S. zeae</i> strains. This molecular method for screening bioactive strains of <i>S. zeae</i> is complementary to other approaches, such as chemical analyses. An <i>e</i>GFP-labelled <i>S. zeae</i> strain was also developed to investigate the endophytic transmission of <i>S. zeae</i> in <i>Z. mays</i> seedlings, which has further improved our understanding of the transmission modes of <i>S. zeae</i> endophytes in maize tissues.