A highly sensitive method for the quantification of methanogens in anaerobic digestor sludges was developed, based on an analysis of ether‐linked glycerolipids. Core lipids were prepared from total lipids by HF treatment and mild methanolysis, and these core lipids were quantified as the corresponding 9‐anthroyl derivatives by high‐performance liquid chromatography with fluorescence detection. The amounts, in terms of cell carbon content, of Methanosaeta and Methanosarcina were proportional to the amounts of α‐hydroxyarchaeol and β‐hydroxyarchaeol, respectively. Moreover, the total amount of core lipids was well correlated with the cell mass of aceticlastic and H2/CO2‐consuming methanogens. The limit of detection for Methanosaeta concilii was 17 ng of cell carbon when the signal/noise ratio was 3. This method allowed us to quantitate aceticlastic methanogens with high accuracy and to make a rough estimate of total methanogenic cells without any interference by the multifarious impurities that are present in anaerobic sludges. These results suggest that the present method will be a useful tool for investigations of methanogenic ecosystems.