Objective: The aim of this study was to examine the frequency and characteristics of patients with gynecologic cancer with microsatellite instability (MSI)-high, as well as the efficacy of pembrolizumab in these patients, by reviewing the experience of a single institution that performs MSI testing. Methods: We retrospectively investigated the frequency and characteristics of MSI-high in patients with advanced or recurrent gynecologic cancer who underwent MSI testing at Toho University Omori Medical Center. As MSI testing, the distribution of microsatellite length at five microsatellite markers (BAT25, BAT26, NR21, NR24, and MONO27) was analyzed by multiplex polymerase chain reaction. We considered to be MSI-high if two or more microsatellite markers were positive. Among patients with MSI-high, we investigated the clinical course of those who received pembrolizumab. Results: MSI testing was performed on 51 patients, four of whom (7.8%) showed MSI-high. Two of the four patients had recurrent endometrial cancer (poorly differentiated endometrioid in one and moderate differentiated endometrioid + clear cell + serous in the other), one had advanced endometrial cancer (stage IVB, poorly differentiated endometrioid), and one had advanced ovarian cancer (stage IIIC, clear cell). No MSI-high was found in the other gynecological cancers. The rate of MSI-high was 15.0% (3/20) for endometrial cancer and 6.7% (1/15) for ovarian cancer. Of the four patients with MSI-high, two received pembrolizumab monotherapy. Both of these patients responded well and have maintained the response for more than 12 months. Discussion: This is the first real-world study investigating MSI-high in Japanese women with gynecologic cancer. MSI testing should be performed aggressively in clinical practice to identify patients with advanced and recurrent gynecologic cancers who can benefit from pembrolizumab.