Background Very few true Pseudomonas methylotrophic strains have been described, and in none of them have the pathways for one-carbon (C 1 ) substrate metabolism been elucidated. Methods The genomes of three Pseudomonas strains able to grow on methanol as the sole source of carbon (C) and energy (E) were sequenced and analyzed, and one of the strains was further characterized at the proteomic and physiological level. Results None of the three strains possesses a classic methanol dehydrogenase enzyme, and they apparently employ generalist type-I alcohol dehydrogenases (ADHs) to catabolize methanol to formaldehyde. In two of the strains’ genomes, the only complete route encoded for incorporating methylotrophic carbon is the Calvin-Benson-Bassham (CBB) cycle, while other more typical pathways for C 1 -carbon assimilation (serine cycle, ribulose monophosphate cycle) appear incomplete. The indispensability of the QedA1 alcohol dehydrogenase and of ribulose bisphosphate carboxylase for growth on methanol was demonstrated by insertion mutagenesis of the qedA1 and cbbL genes in one of the strains. Discussion To the author’s knowledge, all wild-type methylotrophic Pseudomonadota ( i.e. , “Gram-negative bacteria”) so far described employ a specific dehydrogenase distinctively adapted to using methanol as a substrate (MxaFI, XoxFI, or Mdh2). The methylotrophic Pseudomonas strains described here lack MDH and employ generalist ADHs, thus demoting methanol dehydrogenase (MDH) from the position of a critical enzyme for methanol utilization and expanding the range of enzymes (and genes) that enable methylotrophy in nature. The second remarkable result of this work is the discovery of the utilization of the CBB cycle by a Pseudomonas strain during methylotrophic growth, an absolute novelty for this very relevant bacterial genus.