Motile and non‐motile strains of Pseudomonas fluorescens SBW25 were constructed using different combinations of the lacZY, xylE and aph marker genes which allowed their detection and differentiation in soil, root and seed samples. The survival of motile and non‐motile strains was investigated in both non‐competitive and competitive assays in water and non‐sterile soil. Although there was no difference between strains in water, the motile strain survived in significantly greater numbers than the non‐motile strain after 21 days in soil. There was no significant difference between competitive assays, where motile and non‐motile cells were co‐inoculated into soil, and non‐competitive assays where strains were inoculated separately. Bacterial survival decreased as matric potential increased from −224 to −17 kPa but matric potential had no significant effect on motile compared to non‐motile strains. Vertical spread of both motile and non‐motile strains was detected 6.4 mm from the inoculum zone after 14 days in the absence of percolating water. There was no significant difference, for either strain, in distance moved from the inoculum zone after 14, 26 or 40 days. The motile strain had a significant advantage in attachment to sterile wheat roots in both non‐competitive and competitive studies. When the spatial colonisation of wheat root systems was assessed in non‐sterile soil, there was no significant difference between the motile and non‐motile strain from either seed or soil inoculum. However, when the whole root system was assessed as one sample unit, differences could be detected. Bacterial motility could contribute to survival in soil and the initial phase of colonisation, where attachment and movement onto the root surface are important.