Semi-natural grasslands maintained by moderate anthropogenic disturbance have been rapidly declining worldwide in recent decades, resulting in the progressive degradation of biodiversity therein. Similarly, in Japan, many semi-natural grassland organisms are threatened with extinction owing to the decline of semi-natural grasslands, and their conservation and management has attracted considerable attention. Although ecological studies using genetic information are expected to contribute to the conservation of semi-natural grassland organisms, no reviews have been conducted on the potential roles of these studies in grassland conservation in Japan. Here, we review the current trends in conservation-ecological study on Japanese semi-natural grassland organisms using genetic information by grouping into five categories: (1) monitoring of genetic diversity and spatial genetic structure, (2) exploration of the genetic consequences of management practices, (3) prevention of genetic disturbance, (4) elucidation of natural history traits, and (5) estimation of the origin and demography. Furthermore, the applicability of studies using high-throughput sequencing, which has been rapidly progressing in recent years, is highlighted. This review provides the methods and perspectives on the use of genetic information to conserve biodiversity in semi-natural grasslands.