Aims: A molecular tool for extensive detection of prokaryotic alkane hydroxylase genes ( alkB) was developed. AlkB genotypes involved in the degradation of short-chain alkanes were quantified in environmental samples in order to assess their occurrence and ecological importance. Methods and Results: Four primer pairs specific for distinct clusters of alkane hydroxylase genes were designed, allowing amplification of alkB-related genes from all tested alkane-degrading strains and from six of seven microcosms. For the primer pair detecting alkB genes related to the Pseudomonas putida GPo1 alkB gene and the one targeting alkB genes of Gram-positive strains, both involved in short-chain alkane degradation (< C-10), a quantitative competitive PCR (cPCR) assay was developed and validated on alkB-containing strains. AlkB genes of the two groups were then quantified in hydrocarbon-contaminated and pristine freshwater and soil samples, and their respective frequency was compared to degradation rates of short-chain n-alkanes. Pseudomonas putida-related alkB genes were prevalent in freshwater samples, but Gram-positive alkB-containing strains were more consistently related to alkane degradation activities. The latter genotype was more abundant in soils, although both genotypes increased in the most contaminated soils studied. Conclusions: Predominance of alkB genotypes depends on the ecosystem and environmental conditions, but alkane exposure generally leads to an increase of both studied genotypes. Significance and Impact of the Study: The study illustrates the distribution of two different alkB genotypes in two types of ecosystems, and highlights their respective roles in the environment.