Potato Virus Y (PVY) infection and nitrogen (N) deficiency cause similar symptoms (chlorosis and stunting) on potato foliage. While conventional methods, including ELISA and petiole testing, require destructive sampling and a longer time to diagnose, spectral analysis can be non-destructive, rapid and efficient. Spectral reflectance for potato cultivars representing three market types, chip processing, red-skinned fresh, and fresh and processing russets, were assessed in separate greenhouse trials in response to three N rates (90, 200, and 290 kg/ha) and two PVYN:O infection levels (clean and infected) at 4, 6, and 8 weeks after inoculation (WAI). Normalized Difference Vegetation Index (NDVI) was able to differentiate clean and PVYN:O infected samples of red-skinned and chip processing cultivars, at 4 and 8 WAI, respectively. Overall, cultivars differed in their spectral responses, indicating the importance of studying cultivar-specific spectral responses against PVY infection in future.