Setosphaeria turcica, a hemibiotrophic pathogenic dothideomycete, is the causal agent of Northern Leaf Blight of maize, which periodically causes significant yield losses worldwide. To explore molecular mechanisms of fungal pathogenicity and virulence to the host, an efficient targeted gene knockout transformation system using Agrobacterium tumefaciens was established with field collected strains. The starting materials, incubation time, induction medium type, Agrobacterium cell density, and method of co-incubation were optimized for deletion of 1,3,8-trihydroxynaphthalene reductase, a gene in the melanin biosynthesis pathway, as a test case. Four additional genes were deleted in two different S. turcica field isolates to confirm robustness of the method. One of these mutant strains was reduced in virulence compared with the wildtype strain when inoculated on susceptible maize. Transformation efficiency was ≈20 ± 3 transformants per 1 × 106 germlings and homologous recombination efficiency was 33.3 to 100%.