The biosynthetic capability of the bacterial ribosome motivates efforts to understand and harness sequence-optimized versions for synthetic biology. However, functional differences between natively occurring ribosomal RNA (rRNA) operon sequences remain poorly characterized. Here, we use an in vitro ribosome synthesis and translation platform to measure protein production capabilities of ribosomes derived from all unique combinations of 16S and 23S rRNAs from seven distinct Escherichia coli rRNA operon sequences. We observe that polymorphisms that distinguish native E. coli rRNA operons lead to significant functional changes in the resulting ribosomes, ranging from negligible or low gene expression to matching the protein production activity of the standard rRNA operon B sequence. We go on to generate strains expressing single rRNA operons and show that not only do some purified in vivo expressed homogeneous ribosome pools outperform the wild-type, heterogeneous ribosome pool but also that a crude cell lysate made from the strain expressing only operon A ribosomes shows significant yield increases for a panel of medically and industrially relevant proteins. We anticipate that ribosome pool engineering can be applied as a tool to increase yields across many protein biomanufacturing systems, as well as improve basic understanding of ribosome heterogeneity and evolution.