Methylocella spp. are facultative methanotrophs, which are able to grow not only on methane but also on multicarbon substrates such as acetate, pyruvate or malate. Methylocella spp. were previously thought to be restricted to acidic soils such as peatlands, in which they may have a key role in methane oxidation. There is little information on the abundance and distribution of Methylocella spp. in the environment. New primers were designed, and a real-time quantitative PCR method was developed and validated targeting Methylocella mmoX (encoding the a-subunit of the soluble methane monooxygenase) that allowed the quantification of Methylocella spp. in environmental samples. We also developed and validated specific PCR assays, which target 16S rRNA genes of known Methylocella spp. These were used to investigate the distribution of Methylocella spp. in a variety of environmental samples. It was revealed that Methylocella species are widely distributed in nature and not restricted to acidic environments.