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In this work, we applied classical molecular networking analysis to secondary metabolite profiling data from seven\n                      <jats:italic>Pseudomonas aeruginosa<\/jats:italic>\n                      strains, including five clinical isolates from the lung secretions of people with cystic fibrosis (CF). We provide three vignettes illustrating how secondary metabolite profiling aids in the identification of rare genomics traits in\n                      <jats:italic>P. aeruginosa<\/jats:italic>\n                      . First, we describe the identification of a previously unreported class of acyl putrescines produced by isolate mFLRO1. Secondary analysis of publicly available metabolomics data revealed that acyl putrescines are produced by &lt;5% of\n                      <jats:italic>P. aeruginosa<\/jats:italic>\n                      strains. Second, we show that isolate SH3A does not produce di-rhamnolipids. Whole-genome sequencing and comparative genomics revealed that SH3A cannot produce di-rhamnolipids because its genome belongs to clade 5 of the\n                      <jats:italic>P. aeruginosa<\/jats:italic>\n                      phylogenetic tree. Previous phylogenetic analysis of thousands of\n                      <jats:italic>P. aeruginosa<\/jats:italic>\n                      strains concluded that &lt;1% of publicly available genome sequences contribute to this clade. Last, we show that isolate SH1B does not produce the phenazine pyocyanin or rhamnolipids because it has a one-base insertion frameshift mutation (678insC) in the gene\n                      <jats:italic>rhlR<\/jats:italic>\n                      , which disrupts\n                      <jats:italic>rhl<\/jats:italic>\n                      -driven quorum sensing. Secondary analysis of the tens of thousands of publicly available genomes in the National Center for Biotechnology Information (NCBI) and the\n                      <jats:italic>Pseudomonas<\/jats:italic>\n                      Genome Database revealed that this mutation was present in only four\n                      <jats:italic>P. aeruginosa<\/jats:italic>\n                      genomes. Taken together, this study highlights that secondary metabolite profiling combined with genomic analysis can identify rare genetic traits of\n                      <jats:italic>P. aeruginosa<\/jats:italic>\n                      isolates.\n                    <\/jats:p>\n                    <jats:sec>\n                      <jats:title>IMPORTANCE<\/jats:title>\n                      <jats:p>\n                        Secondary metabolite profiling of five\n                        <jats:italic>Pseudomonas aeruginosa<\/jats:italic>\n                        isolates from cystic fibrosis sputum captured three traits present in &lt;1%\u20135% of publicly available data, pointing to how our current library of\n                        <jats:italic>P. aeruginosa<\/jats:italic>\n                        strains may not represent the diversity within this species or the genetic variance that occurs in the CF lung.\n                      <\/jats:p>\n                    <\/jats:sec>\n                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