Chlorophyll fluorescence (CF) measurements have been demonstrated to be an efficient and non-invasive tool for identifying and developing PVY-resistant potato cultivars. The validity of CF measurements was confirmed through viral titer and yield-loss assays. In the quest to identify resistant sources for potato virus Y (PVY) within Indian potato germplasm, we developed a phenotyping approach leveraging plant physiological responses against PVY infection. The study evaluated 71 potato genotypes including cultivated and experimental clones, during the year 2021-2022 and 2022-23 through mechanical inoculation in experimental fields at the Punjab Agricultural University, Ludhiana. We employed a combination of serological and molecular screening, complemented with chlorophyll fluorescence (CF) measurements to classify resistant and susceptible genotypes. Out of 71 genotypes, 34 exhibited PVY resistance, with KP-16-19-14 being the highly resistant line with minimal yield loss (i.e., only 1.64% reduction) and undetectable viral titer. This genotype holds promise as a valuable resistance source for future breeding programmes. Our findings revealed that resistant genotypes maintained stable CF metrics and experienced minimal yield reductions (up to 5.15% only), with very low viral titer. In contrast, the photosynthetic efficiency was significantly declined in susceptible genotypes, which also experienced yield losses up to 58.84% with very high viral titer. Correlation coefficient and principal component analysis (PCA) revealed a strong association among the CF parameters, disease severity, viral titer, and yield losses. This emphasizes the utility of CF as a valuable tool for assessing resistance through physiological responses to PVY. Study demonstrates that photochemistry, heat dissipation, and fluorescence emission patterns of PS-II effectively differentiate resistant and susceptible genotypes. Moreover, this study highlights the potential of integrating physiological assessments with molecular diagnostics in large-scale preliminary screening to identify and develop PVY-resistant potato genotypes.