Colony morphology variants isolated from natural and laboratory-grown biofilms represent subpopulations of biofilm cells that may be important for multiple aspects of the sessile lifestyle, from surface colonization to stress resistance. There are many genetic and environmental factors that determine the frequency at which colony morphology variants are recovered from biofilms. One of these factors involves an increased selection for variants in biofilms of Pseudomonas species bearing inactivating mutations in the global activator of cyanide biosynthesis/regulator of secondary metabolism (gac/rsm) signal transduction pathway. Here we characterize two distinct colony morphology variants isolated from biofilms of Pseudomonas fluorescens missing the gacS sensor kinase. These variants produced more biofilm cell mass, and in one case, this was likely due to overproduction of the exopolysaccharide cellulose. Nuclear magnetic resonance (NMR) metabolomics revealed distinct metabolic changes for each of the two phenotypic variants, and these changes involved amino acids and metabolites produced through glutathione biochemistry. Some of these metabolites are hypothesized to play a role in redox and metal homeostasis, and corresponding to this, we show that biofilm populations grown from each of these variants had a different ability to survive when exposed to toxic doses of metal ions. These data suggest that colony morphology variants that evolve during growth of P. fluorescens as a biofilm may have distinct metabolic capacities that contribute to their individual abilities to withstand environmental stress.