Genetic improvement of grapevine for resistance against downy mildew (Plasmopara viticola) by biotechnological techniques requires reliable procedures to screen large populations of plants. In comparison with greenhouse-grown plants, in vitro plantlets are not often used in screening procedures, although they present some advantages such as compact size, the availability of a high number of replicates per genotype, and the potential to screen improved genotypes directly from in vitro plants. Leaf disc inoculation bioassay was used to evaluate grapevine resistance to downy mildew on Vitis vinifera Chardonnay, hybrid Seyval, and Vitis riparia Gloire de Montpellier (susceptible, moderately susceptible, and highly resistant, respectively) with both in vitro plantlets and greenhouse-grown plants. Disease symptoms and resistance reaction were evaluated for sporulation and necrosis in two independent experiments. For all parameters, leaves from in vitro plantlets appeared more resistant than leaves from greenhouse-grown plants, in particular for the intermediate and the susceptible genotypes. Necroses were not observed on leaf discs of the susceptible genotype, whereas necrotic spots appeared on leaf discs of both intermediate and resistant genotypes regardless of how the plants were grown. Based on sporulation and necrosis symptoms, ranking of genotypes differed according to growing conditions. Although the method based on in vitro grown plants is less reliable than that based on greenhouse plants, it can be used as a preliminary assay to eliminate the most susceptible plantlets obtained by biotechnology.