Aztreonam is a relatively new antibiotic which is being increasingly used to treat cystic fibrosis patients with Pseudomonas aeruginosa airway infections. As for all antimicrobial agents, bacteria can evolve resistance and decrease the effectiveness of the drug; however, the mechanisms and consequences of aztreonam resistance are incompletely understood. Using experimental evolution, we have cataloged spontaneous mutations conferring aztreonam resistance and have explored their effects on bacterial fitness, the potential for cross resistance, and virulence within a host.