Hypersensitive response (HR) of plant cells to the attack of pathogens induces resistance to subsequent attacks by a broad spectrum of pathogens, leading to acquired resistance. In this study, we characterized the localized acquired resistance (LAR) in the epidermal cells of tomato. First, we report the discovery of a new isolate of tomato powdery mildew occurring in Japan, KTP-02, which has a different virulence spectrum compared with the previously-characterized isolate, KTP-01. Using these two isolates, we investigated LAR phenomenon in the epidermal cells of tomato plants carrying the Ol-4 resistance gene. Ol-4 encodes a nucleotide-binding site leucine-rich repeat protein that triggers HR in the epidermal cells in response to KTP-01 but not KTP-02. We mounted a single conidium of KTP-01 on a single tomato epidermal cell and then monitored the progress of HR in that cell by live microscopy. Once HR occurred in that cell, we mounted a single conidium of KTP-02 on cells adjacent to or at one-cell distance from the first challenged cells, in different time points. With a digital microscope, we consecutively tracked the progress of HR (i.e., induction of LAR) in those cells. Results showed that, in tomato plants carrying the Ol-4 gene, HR to KTP-01 results in induction of HR in the adjacent epidermal cells challenged with KTP-02. Our results show that LAR can be triggered only in adjacent cell layer and lasts 24 to 48 h after HR occurred in the first cell. We did not observe the reverse phenomenon, induced susceptibility to KTP-01 by KTP-02. Altogether, we report an advanced technique for investigating LAR phenomena, and provide data on spatiotemporal characteristics of LAR in tomato epidermal cells.