Fusarium verticillioides (teleomorph Gibberella moniliformis) is associated with maize worldwide causing ear rot and stalk rot, and produces fumonisins, a group of mycotoxins detrimental to humans and animals. While research tools are available, our understanding of the molecular mechanisms associated with fungal virulence and fumonisin biosynthesis in F. verticillioides is still limited. One of the restraints that hampers F. verticillioides gene characterization is the fact that homologous recombination (HR) frequency is very low (<2%). Screening for a true gene knock-out mutant is a laborious process due to a high number of ectopic integrations. In this study, we generated a F. verticillioides mutant (SF41) deleted for FvKU70, a gene directly responsible for non-homologous end-joining mechanism, with the aim of improving HR frequency. Here, we demonstrate that FvKU70 deletion does not impact key F. verticillioides phenotypes, e.g., development, secondary metabolism, and virulence, while dramatically improving HR frequency. Significantly, we also confirmed that a high percentage (>85%) of the HR mutant strains harbor a desired mutation with no additional copy of the mutant allele inserted in the genome. We conclude that SF41 is suitable for use as a type strain when performing high-throughput gene function studies in F. verticillioides.