This study aimed to define progesterone 5β-reductases (P5βR, EC 1.3.99.6, enone 1,4-reductases) as function-associated molecular markers at the plant family level. Therefore cDNAs were isolated from 25 Brassicaceae species, including two species, Erysimum crepidifolium and Draba aizoides, known to produce cardiac glycosides. The sequences were used in a molecular phylogeny study. The cladogram created is congruent to the existing molecular analyses. Recombinant His-tagged forms of the P5βR cDNAs from Aethionema grandiflorum, Draba aizoides, Nasturtium officinale, Raphanus sativus and Sisymbrium officinale were expressed in E. coli. Enone 1,4-reductase activity was demonstrated in vitro using progesterone and 2-cyclohexen-1-one as substrates. Evidence is provided that functional P5βRs are ubiquitous in the Brassicaceae. The recombinant P5βR enzymes showed different substrate preferences towards progesterone and 2-cyclohexen-1-one. Sequence comparison of the catalytic pocket of the P5βR enzymes and homology modelling using Digitalis lanata P5βR (PDB ID: 2V6G) as template highlighted the importance of the hydrophobicity of the binding pocket for substrate discrimination. It is concluded that P5βR genes or P5βR proteins can be used as valuable function-associated molecular markers to infer taxonomic relationship and evolutionary diversification from a metabolic/catalytic perspective.