The desotamides (DSAs) are potent antibacterial cyclohexapeptides produced by Streptomyces scopuliridis SCSIO ZJ46. We have identified the 39-kb dsa biosynthetic gene cluster by whole-genome scanning. Composed of 17 open reading frames, the cluster codes for four nonribosomal peptide synthetases and associated resistance, transport, regulatory, and precursor biosynthesis proteins. Heterologous expression of the dsa gene cluster in S. coelicolor M1152 afforded desotamides A and B and the new desotamide G. Cluster identification and its demonstrated amenability to heterologous expression provide the foundation for future mechanistic studies as well as the generation of new and potentially clinically significant DSA analogues.