Abstract Rare plant species identified in project areas where land is to be developed or altered may need to be extracted for ex situ conservation under human control. During ex situ conservation, it is important to minimize the impact of adverse factors including the potentially limited environmental adaptability of rare plants under cultivation and possible losses of genetic diversity due to inbreeding if the number of conserved individuals is low. Here we evaluate the conservation difficulty and genetic diversity of populations of the rare plant Violα raddeana at five sites based on harmful gene accumulation and gene duplication data gathered using RNA‐seq. Additionally, we evaluate the genetic diversity and structure of these populations using single‐nucleotide polymorphism (SNP) data acquired by multiplexed inter‐simple sequence repeat genotyping by sequencing (MIG‐seq). The results obtained indicate that the V. raddeana populations have declined but remain robust toward environmental changes and can undergo voluntary mating at all sites. Moreover, we observed clear genetic differentiation between two populations in close geographic proximity. We considered that if the majority of the remaining individuals of V. raddeana in each genetically related population were transplanted and cultivated, it would be possible to maintain their genetic diversity through ex situ conservation. It is important that V. raddeana be kept under ex situ conservation management. In order to achieve this, we should work with public organizations such as local government agencies and botanical gardens.