The evolutionary success of the novel Wrinkly Spreader (WS) genotypes in diversifying Pseudomonas fluorescens SBW25 populations in static liquid microcosms has been attributed to the greater availability of O2 at the air–liquid (A–L) interface where the WS produces a physically cohesive-class biofilm. However, the importance of O2 gradients in SBW25 adaptation has never been examined. We have explicitly tested the role of O2 in evolving populations using microsensor profiling and experiments conducted under high and low O2 conditions. Initial colonists of static microcosms were found to establish O2 gradients before significant population growth had occurred, converting a previously homogenous environment into one containing a resource continuum with high and low O2 regions. These gradients were found to persist for long periods by which time significant numbers of WS had appeared colonising the high O2 niches. Growth was O2 limited in static microcosms, but high O2 conditions like those found near the A–L interface supported greater growth and favoured the emergence of WS-like genotypes. A fitness advantage to biofilm formation was seen under high but not low O2 conditions, suggesting that the cost of biofilm production could only be offset when O2 levels above the A–L interface were high. Profiling of mature WS biofilms showed that they also contained high and low O2 regions. Niches within these may support further diversification and succession of the developing biofilm population. O2 availability has been found to be a major factor underlying the evolutionary success of the WS genotype in static microcosms and illustrates the importance of this resource continuum in microbial diversification and adaptation.