Streptomyces rimosus, the industrial strain used in the production of the Type-II polyketide antibiotic oxytetracycline (OTC), has undergone extensive strain improvement over the past 50 years. This has resulted in OTC levels increasing from less than 0.5 g per litre in the original soil isolate to over 70 g per litre for contemporary production strains. By analysing the genome sequences of four strains that lie within this lineage, each with a different level of OTC productivity, we have investigated the genetic basis of increased OTC yield. Using comparative genomics, a variety of significant chromosomal rearrangements and deletions in the lineage have been identified. These chromosomal rearrangements have resulted in a loss of genes involved in primary metabolism, the synthesis of other secondary metabolites, transcriptional regulators and sigma factors. Most notable, is the loss of the rimocidin biosynthetic cluster in later strains, which suggests that increased precursor supply is an important determinant for increased OTC yield. To confirm this association and to elucidate the significance of the observed genomic rearrangements, gene knockouts were produced in the original soil isolate. A transcriptional regulator putatively associated with the OTC biosynthetic gene cluster was also identified during this analysis. Further to this, the regulator in the one of the production strains contained a mutation within the DNA-binding region of the protein. It is possible this mutation alters DNA-binding affinity and therefore improves OTC production. Ultimately, we aim to use our genomic data to inform rapid improvement of other strains.