The ability of bacteria to alter phenotypic characteristics during their survival and persistence in soil complicates the risk assessment of bacterial inoculants, especially those genetically engineered. The purpose of this study was to investigate changes in the phenotype of Pseudomonas aureofaciens 3732 RN-L11 (lacZY) introduced into and recovered from wheat roots or soil obtained from the field or laboratory microcosms. Fatty acid methyl ester (FAME) and Biolog™ analyses were used to characterize the phenotype of 88 isolates obtained from soil collected over a 2-year period. There was variation in the relative proportions of fatty acids found in isolates over the growing season, with FAME profiles of isolates obtained at 14, 28 and 70 days after planting different from isolates obtained at day 0 and day 140. However, these changes were not sufficient to alter the species designation. Similarly, carbon substrate utilization by isolates varied only slightly over the growing season. In addition, there were few phenotypic differences found between isolates obtained from rhizosphere or bulk soil, and no observed differences between isolates recovered from field soil or microcosms. Our results indicate that there was little phenotypic drift of this genetically engineered bacterium during its survival in field and laboratory microcosm soils.