Cyclic cysteine-rich plant peptides (cyclotides) are found in many plant species of the Rubiaceae, Fabaceae, Violaceae, Cucurbitaceae, Solanaceae and Poaceae families. Despite promising applications as drug templates, little is understood in cyclotide’s distribution, evolution and variability in plants and of in planta bioprocessing. Widely recognised identification criteria in peptidomics for cyclotide research are their strong retention in reversed phase chromatography, a molecular mass of 2500-4000 Da, a head-to-tail cyclized backbone and a knotted arrangement of six conserved cysteines, nevertheless a valid identification relies on de novo sequencing of at least two adjacent inter-cysteine loops of the peptide1. However, cyclotide screening is prone to false-negative or false-positive hits. Hence, more robust criteria for large scale screening studies are of interest in cyclotide research2. Here we present a combined peptidomics/transcriptomics approach to overcome limitations in conventional cyclotide discovery3. Applying this refined identification workflow, ~300 species of >40 plant families with focus on Rubiaceae species were evaluated. Altogether six full sequences, ten partial sequence tags and nine new cyclotide expressing families all from the Palicoureeae tribe were identified for the first time. Two novel sequences from Carapichea ipecacuanha lack of the highly conserved glutamic acid, thus our finding underlines the little understood plasticity of these plant peptides. In particular transcriptome mining provides the full primary sequence and eliminates false-negative hits. Besides phylogenetic insight into cyclotides distribution in Psychotria species, comparison of cyclotide precursor sequences amongst the Rubiaceae family revealed a moderate variability for several amino acid residues on the proto-N and proto-C-termini previously described as highly conserved due to their involvement in in planta bioprocessing. In conclusion, combined peptidomics/transcriptome analysis is a powerful tool to shed light on the distribution, evolution and diversity of circular, disulfide-rich peptides within the plant kingdom and enables targeted screening to accelerate peptide discovery.