Non-ribosomal peptide synthetase (NRPS) is a diverse family of biosynthetic enzymes for the assembly of bioactive peptides. Despite advances in microbial sequencing, the lack of a consistent standard for annotating NRPS domains and modules has made data-driven discoveries challenging. To address this, we introduced a standardized architecture for NRPS, by using known conserved motifs to partition typical domains. This motif-and-intermotif standardization allowed for systematic evaluations of sequence properties from a large number of NRPS pathways, resulting in the most comprehensive cross-kingdom C domain subtype classifications to date, as well as the discovery and experimental validation of novel conserved motifs with functional significance. Furthermore, our coevolution analysis revealed important barriers associated with re-engineering NRPSs and uncovered the entanglement between phylogeny and substrate specificity in NRPS sequences. Our findings provide a comprehensive and statistically insightful analysis of NRPS sequences, opening avenues for future data-driven discoveries.