Background: Cardamine flexuosa is considered to be two separate species in the Cardamine genus based on their geographical distribution: European C. flexuosa and Eastern Asian C. flexuosa. These two species have not shown any morphological differences to distinguish each other. Recently, the Eastern Asian species has been regarded as Cardamine occulta by their ecological habitats. Therefore, we are interested in analyzing the C. occulta chloroplast genome and its characteristics at the molecular level. Methods: Here, the complete chloroplast (cp) genome of C. occulta was assembled de novo with next-generation sequencing technology and various bioinformatics tools applied for comparative studies. Results: The C. occulta cp genome had a quadripartite structure, 154,796 bp in size, consisting of one large single-copy region of 83,836 bp and one small single-copy region of 17,936 bp, separated by two inverted repeats (IRa and IRb) regions of 26,512 bp. This complete cp genome harbored 113 unique genes, including 80 protein-coding genes, 29 tRNA, and four rRNA genes. Of these, six PCGs, eight tRNA, and four rRNA genes were duplicated in the IR region, and one gene, infA, was a pseudogene. Comparative analysis showed that all the species of Cardamine encoded a small variable number of repeats and SSRs in their cp genome. In addition, 56 divergences (Pi > 0.03) were found in the coding (Pi > 0.03) and non-coding (Pi > 0.10) regions. Furthermore, KA/KS nucleotide substitution analysis indicated that thirteen protein-coding genes are highly diverged and identified 29 amino acid sites under potentially positive selection in these genes. Phylogenetic analyses suggested that C. occulta has a closer genetic relationship to C. fallax with a strong bootstrap value. Conclusions: The identified hotspot regions could be helpful in developing molecular genetic markers for resolving the phylogenetic relationships and species validation of the controversial Cardamine clade.