Maize yield is threatened by increasing incidences of head smut disease caused by Sporisorium reilianum. To help breeders identify S. reilianum-resistant maize lines, the availability of efficient screening systems would be an advantage. Here we assessed maize lines with distinct levels of field resistance against head smut disease in greenhouse experiments using two different inoculation techniques. Addition of mixtures of mating-compatible sporidia to the soil at seedling stage of the plant did not lead to plant disease, and we could detect only marginal amounts of fungal DNA in apical meristems at eighteen days after sowing. Inoculation of the maize lines by leaf-whorl inoculation led to both high disease incidence and prominent levels of fungal DNA in apical meristems in all tested maize lines regardless of their field resistance levels. Thus, S. reilianum entering the plant via the leaf whorl can escape existing resistance mechanisms of currently known field-resistant maize lines. Since field-resistant lines are also resistant to inoculation via teliospore-contaminated soil, we propose teliospore addition to seeds at the time of sowing (rather than leaf-whorl inoculation of seedlings) combined with quantitative detection of fungal DNA in apical meristems, as an efficient screening procedure to discover field-resistant lines. However, screening maize plants for resistance against the leaf-whorl inoculation method might be promising for the discovery of novel resistance mechanisms needed to develop durably resistant maize lines.