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Summary  A form of lateral membrane compartmentalization in bacteria is represented by functional membrane microdomains (FMMs). FMMs are important for various cellular processes and offer application possibilities in microbial biotechnology. We designed a lipidomics method to directly measure relative abundances of lipids in detergent‐resistant and detergent‐sensitive membrane fractions of the model bacterium  Bacillus subtilis  168 and the biotechnologically attractive mini Bacillus  PG10 strain. Our study supports previous work suggesting that cardiolipin and prenol lipids are enriched in FMMs of  B. subtilis . Additionally, structural analysis of acyl chains of major phospholipids indicated that FMMs display increased order and thickness compared with the surrounding bilayer. Despite the 36% genome reduction, membrane and FMM integrity are largely preserved in mini Bacillus  PG10, as supported by analysis of membrane fluidity, flotillin distribution and gene expression data. The novel insights in FMM architecture reported here will contribute to further explore the biological significance of FMMs and the means by which FMMs can be exploited as heterologous production platforms. Moreover, our lipidomics method enables comparative FMM lipid profiling between different bacteria.

Membrane composition and organization of Bacillus subtilis 168 and its genome‐reduced derivative mini Bacillus PG10