The type strains of 32 species of 13 genera of the family Microbacteriaceae were analysed with respect to gene-coding phylogeny for DNA gyrase subunit B (gyrB), RNA-polymerase subunit B (rpoB), recombinase A (recA), and polyphosphate kinase (ppk). The resulting gene trees were compared with the 16S rRNA gene phylogeny of the same strains. The topology of neighbour-joining and maximum parsimony phylogenetic trees, based on nucleic-acid sequences and protein sequences of housekeeping genes, differed from one another, and no gene tree was identical to that of the 16S rRNA gene tree. Most genera analysed containing > 1 strain formed phylogenetically coherent taxa. The three pathovars of Curtobacterium flaccumfaciens clustered together to the exclusion of the type strains of other Curtobacterium species in all DNA - and protein-based analyses. In no tree did the distribution of a major taxonomic marker, i.e., diaminobutyric acid versus lysine and/or ornithine in the peptidoglycan, or acyl type of peptidoglycan, correlate with the phylogenetic position of the organisms. The changing phylogenetic position of Agrococcus jenensis was unexpected: This strain defined individual lineages in the trees based on 16S rRNA and gyrB and showed identity with Microbacterium saperdae in the other three gene trees.