The heathland lichen Cladonia portentosa was collected from sites in mainland Britain differing either in rates of wet N deposition or in annual mean N concentration in rainfall based on a modelled data set. Methanolic extracts of thalli were analyzed by liquid chromatography time-of-flight mass spectrometry to yield metabolic profiles. Differences between sites in metabolite concentration were quantified using multivariate statistical tools and used to identify potential biomarker molecules. The abundances of three structurally related betaine lipids showed an increase with increasing modelled N deposition to a threshold of 22.3 kg ha-1 year-1 after which they remained constant. In contrast, the abundance of a phosphatidylcholine (PC) lipid showed concomitant decrease. Correlations of the identified biomarkers with N deposition and precipitation were stronger than those with N concentrations. The results presented in this study clearly show that N enrichment associated with tissue P limitation changes lipid composition, leading to shifts from PCs to betaine lipids, and that these lipids identified have the potential to be used as biomarkers for nitrogen enrichment.