Bacillus methanolicus can utilize methanol as its sole carbon and energy source and the scientific interest in this thermotolerant bacterium has largely focused on exploring its potential as a biocatalyst for conversion of methanol into l-lysine and l-glutamate. We here present the genome sequences of the important B. methanolicus model strain MGA3 (ATCC53907) and the alternative wild-type strain PB1 (NCIMB13113). The physiological diversity of these two strains was demonstrated by a comparative fed-batch methanol cultivation displaying highly different methanol consumption and respiration profiles, and also major differences in their l-glutamate production levels (406 mmol l-1 and 11 mmol l-1, respectively). Both genomes are small (ca 3.4 Mbp) compared to other related bacilli, and MGA3 has two plasmids (pBM19 and pBM69) while PB1 has only one (pBM20). In particular, we here focus on genes representing biochemical pathways for methanol oxidation and concomitant formaldehyde assimilation and dissimilation, the important phosphoenol pyruvate/pyruvate anaplerotic node, the tricarboxylic acid cycle including the glyoxylate pathway, and the biosynthetic pathways for l-lysine and l-glutamate. Several unique findings were made including the discovery of three different methanol dehydrogenase genes in each of the two B. methanolicus strains, and the genomic analyses were accompanied with gene expression studies. Our results provide new insight into a number of peculiar physiological and metabolic traits of B. methanolicus and open for system-level metabolic engineering of this bacterium for the production of amino acids and other useful compounds from methanol.