Lithoautotrophic nitrite-oxidizing bacterial populations from moving-bed biofilters of brackish recirculation aquaculture systems (RAS; shrimp and barramundi) were tested for their metabolic activity and phylogenetic diversity. Samples from the biofilters were labeled with (13)C-bicarbonate and supplemented with nitrite at concentrations of 0.3, 3 and 10 mM, and incubated at 17 and 28°C, respectively. The biofilm material was analyzed by fatty acid methyl ester - stable isotope probing (FAME-SIP). High portions of up to 45% of Nitrospira-related labeled lipid markers were found confirming that Nitrospira is the major autotrophic nitrite oxidizer in these brackish systems with high nitrogen loads. Other nitrite-oxidizing bacteria such as Nitrobacter or Nitrotoga were functionally not relevant in the investigated biofilters. Nitrospira-related 16S rRNA gene sequences were obtained from the samples with 10 mM nitrite and analyzed by a cloning approach. Sequence studies revealed four different phylogenetic clusters within the marine sublineage IV of Nitrospira, though most sequences clustered with the type strain of Nitrospira marina and with a strain isolated from a marine RAS. Three lipids dominated the whole fatty acid profiles of nitrite-oxidizing marine and brackish enrichments of Nitrospira sublineage IV organisms. The membranes included two marker lipids (16∶1 cis7 and 16∶1 cis11) combined with the non-specific acid 16∶0 as major compounds and confirmed these marker lipids as characteristic for sublineage IV species. The predominant labeling of these characteristic fatty acids and the phylogenetic sequence analyses of the marine Nitrospira sublineage IV identified organisms of this sublineage as main autotrophic nitrite-oxidizers in the investigated brackish biofilter systems.