This study was conducted to interpret the differences in frost tolerance and winter survival between 8x, 10x, 12x, 14x, and 16x ploidy levels of garden pansy (Viola × wittrockiana Gams) genotypes grown in the field conditions. Plants of each genotype were analyzed for their ploidy levels using flow cytometry. The chlorophyll fluorescence parameters were measured with portable chlorophyll fluorometer in the greenhouse and in the field at different time intervals. Increased frost stress generally reduced the fluorescence values in all genotypes. However, the genotypes differed significantly in their responses to frost as they were exposed to minimum temperatures of 1°C to –7.7°C in the field. Based on the percentage reduction in F_V/F_M values against –7.7°C temperature the hexadecaploids were ranked as sensitive to intermediate followed by 12x (sensitive), and genotypes with 10x and 14x ploidy levels were tolerant as the controls. The winter survival rate of hexadecaploids was by 7 to 9% lower than in the controls followed by the genotype with 12x and both genotypes with 10x and 14x ploidy levels were about equal to the controls. On the other hand, the content of photosynthetic pigments (chlorophyll a, b and total carotenoids) was the highest in hexadecaploids and tended to increase with increasing ploidy level. Further, the results gave insight that chlorophyll fluorescence could be applied directly in the field conditions to screen genotypes and select plants having higher frost tolerance in combination with improved aesthetic qualities.