Ribosomally produced natural products, the RiPPs, exhibit features that are potentially useful in the creation of large chemical libraries using simple mutagenesis. RiPPs are encoded on ribosomal precursor peptides, but they are extensively posttranslationally modified, endowing them with properties that are useful in drug discovery and biotechnology. In order to determine which mutations are acceptable, strategies are required to determine sequence selectivity independently of the context of flanking amino acids. Here, we examined the absolute sequence selectivity of the trunkamide cyanobactin pathway, tru. A series of random double and quadruple simultaneous mutants were synthesized and produced in Escherichia coli. Out of a total of 763 mutated amino acids examined in 325 unique sequences, 323 amino acids were successfully incorporated in 159 sequences, leading to >300 new compounds. Rules for tru sequence selectivity were determined, which will be useful for the design and synthesis of combinatorial biosynthetic libraries. The results are also interpreted in comparison to the known natural products of tru and pat cyanobactin pathways.