Polyketides are an important group of natural products, due to their pharmaceutical applications. Enzymes responsible for the production of polyketides are widely distributed in nature and are produced by plants, bacteria and fungi. One group of particularly interesting enzymes is chalcone synthases (CHS), first identified in plants. This group of enzymes are responsible for the initial step in flavonoid biosynthesis. In bacteria, similar enzymes have been identified and shown to have antitumor and antiplasmodial activity. To understand the similarities between plant and CHSs from other organisms, we have carried out a phylogenetic analysis of plant, bacterial and fungal synthases and biochemically characterized a cyanobacterial chalcone-like synthase. The phylogeny showed that plant CHSs have evolved from cyanobacteria, specifically from the filamentous and nitrogen-fixing order Nostocales, suggestive of a similar reaction mechanism between plants and cyanobacteria. Comparison of the active site between plant and CHS-like enzymes from Nostocales showed structural similarities and are unlike the active sites from bacteria and fungi. Surprisingly, the analysis found that cyanobacterial chalcone-like synthases have evolved twice – once within a hybrid cluster (Type I/III polyketide synthase systems) and once as a single enzyme cluster. Using E. coli as a host, we have cloned and heterologously expressed a single CHS-like enzyme from the cyanobacterium Raphidopsis mediterranea (order Nostocales). Using several approaches (bioinformatics, HPLC and NMR), we have characterized its product. Comparisons to the plant CHSs shows several differences in their substrate specificity and end-product, which broadens our knowledge of this interesting enzyme group, both in plants and in microorganisms.