Stacking resistance genes has been suggested as a path to durable resistance. For the Vitis / Erysiphe necator pathosystem, we assessed the impact of two major (RUN1 and REN6) and two moderate (REN1 and REN7) resistance loci combined in 16 unique full-sib host genotypes (verified by AmpSeq markers). Each genotype was inoculated using 3 E. necator isolates with differential virulence on RUN1 vines (avirulent NY90, fully virulent Musc4, and moderately virulent NY1-137). Disease progress was assessed non-destructively at 2, 4, 6 and 9 days post inoculation using a high-throughput robotic system and convolutional neural network, verified by manual microscopic examinations. Disease severity increased as a linear or exponential function over time in all genetic backgrounds. These preliminary data suggest that stacking a second gene with REN1 or REN7 provided more resistance than REN1 or REN7 alone, but in contrast RUN1 or REN6 resistance was not significantly improved when more genes were added. An exception was observed with Musc4, which coevolved with V. rotundifolia and is fully virulent on RUN1 vines; it colonized more tissue than the other isolates on RUN1-containing germplasm. NY1-137, which was recently isolated from RUN1 vines, was intermediate in virulence. The foregoing results suggest that efficacy and durability of gene stacking depends on the resistance genes used and the virulence carrying capacity of the pathogen.