We demonstrate temporal-, spatial- and temperature-dependent expression of five host microRNAs and target mRNAs in potato leaves after potato virus Y (PVY) inoculation. A novel gene, Ny-DG, conferring resistance to PVY was mapped on chromosome IX in the diploid potato clone DG 81-68. At 20 °C, the PVYNTN strain was localized in the inoculated leaves without symptoms. At 28 °C, necrotic lesions were observed in both the inoculated leaves and the non-inoculated upper leaves and the virus spread systemically, which indicates overcoming the resistance. At 0.5 h post-inoculation and 3-day post-inoculation (dpi), PVY led to significant changes in the levels of the tested miRNAs and their targets only in a small number of cases. At 6 dpi, in the inoculated leaves at 28 °C, the increased expression of stu-miR162, stu-miR168a and stu-miR482 (PotatoMir1005353123_x15854) promoted the downregulation of their targets DCL1, AGO1-2 and Cc-nbs-lrr, respectively. The expression of stu-miR482 (PotatoMir1005658171_x16170) and stu-miR172e remained unchanged, whereas their targets Gpa2 and TOE3 were downregulated. However, in the inoculated leaves at 20 °C, all five tested miRNAs and their targets showed parallel downregulation. In the non-inoculated upper leaves, changes in gene expression levels, namely downregulation, were only detected for the tested five miRNAs at 28 °C. We conclude that the expression patterns of the tested miRNAs and targets were altered differently not only at different time points post-inoculation but also in the inoculated and upper leaves, and their accumulation levels were related to the type of reaction, which in turn was dependent on temperature.