Diketopiperazines (DKPs) are derivatives of cyclodipeptides resulted from the condensation of two amino acids. The conformationally constrained six-membered ring makes DKP an attractive pharmacophore in medicinal chemistry. Recently, there has been increasing interest in the natural DKPs due to their diverse bioactivities and pharmacological activities. Advances in modern biotechnologies and high throughput sequencing technologies have promoted our understanding of DKPs biosynthesis both on enzymatic and genetic levels. Biosynthesis of DKPs can be achieved either by non-ribosomal peptide synthases (NRPSs) or cycliodipeptide synthases (CDPSs). This paper provides a brief overview of recent progresses on DKPs biosynthesis.