ABSTRACT Bacillus subtilis has been extensively used to study the molecular mechanisms behind the development and dispersal of surface bacterial multicellular communities. Well-structured spatially organised communities (colony, pellicle, and submerged biofilm) share some similarities, but also display considerable differences at the structural, chemical and biological levels. To unveil the spatial transcriptional heterogeneity between the different communities, we analysed by RNA-seq nine spatio-physiological populations selected from planktonic and spatially organised communities. This led to a global landscape characterisation of gene expression profiles uncovering genes specifically expressed in each compartmental population. From this mesoscale analysis and using fluorescent transcriptional reporter fusions, 17 genes were selected and their patterns of expression reported at single cell scale with time-lapse confocal laser scanning microscopy (CLSM). Derived kymographs allowed to emphasise spectacular mosaic gene expression patterns within a biofilm. A special emphasis on oppositely regulated carbon metabolism genes ( gapA and gapB ) permitted to pinpoint the coexistence of spatially segregated bacteria under either glycolytic or gluconeogenic regime in a same biofilm population. Altogether, this study gives novel insights on the development and dispersal of B. subtilis surface-associated communities.