Karyotypic changes in chromosome number and structure are drivers in the divergent evolution of diverse plant species and lineages. This study aimed to reveal the origins of the unique karyotype (2n = 12) and phylogenetic relationships of the genus Megadenia (Brassicaceae). A high-quality chromosome-scale genome was assembled for Megadenia pygmaea using Nanopore long reads and high-throughput chromosome conformation capture (Hi-C). The assembled genome is 215.2-Mb and is anchored on six pseudo-chromosomes. We annotated a total of 25,607 high-confidence protein-coding genes and corroborated the phylogenetic affinity of Megadenia with the expanded Lineage II, which contains numerous agricultural crops. We dated the divergence of Megadenia from its closest relatives to 27.04 (19.11-36.60) million years ago. A reconstruction of the chromosomal composition of the species was performed based on the de novo assembled genome and comparative chromosome painting analysis. The karyotype structure of M. pygmaea is very similar to the previously inferred Proto-Calepineae Karyotype (PCK; n = 7) of the Brassicaceae Lineage II. However, an end-to-end translocation between two ancestral chromosomes reduced the chromosome number from n = 7 to n = 6, comparable to Megadenia. Our reference genome provides fundamental information for use in horticulture, plant breeding and evolutionary study of this genus.