Biomarkers will play important roles in disease diagnosis, drug development, and the proper use of drugs. Blood is considered the best biofluid for biomarker research because it is easy to access and a wealth of data are available. However, previous studies revealed that several ionic metabolites showed different levels (including presence or absence) in plasma and serum. Thus, attention should be paid to selecting the best biofluid for biomarker exploration. Many lipid molecules have biological significance and thus would be candidate biomarkers. However, no comprehensive study revealing differences in lipid metabolite levels between plasma and serum has been undertaken. Furthermore, gender differences have not been reported. To clarify the difference in the levels of lipid metabolites between human plasma and serum from both genders, we performed lipid metabolomic analysis using liquid chromatography-mass spectrometry-based systems for phospholipids (PLs), lysoPLs, sphingomyelins, ceramides and oxidative fatty acids. Our results revealed that most of the lipid metabolites were present at similar levels in plasma and serum and in males and females. However, several oxidative fatty acid metabolites showed differences. Of the metabolites related to clotting processes, three showed higher levels in serum than in plasma, and three were detected only in serum. Furthermore, four metabolites were present at different levels between males and females, and two were detected only in males. Thus, attention should be paid to the selection of plasma or serum when utilizing these lipid metabolites as biomarkers.