Mycoplasma pneumoniae expresses β-glycolipids (β-GGLs) in cytoplasmic membranes, which possess a unique β(1 → 6)-linked disaccharide epitope, which has high potential in biochemical and medicinal applications. In the present study, a series of β-GGLs homologues with different acyl chains (C12, C14, C16, and C18) were prepared from a common precursor. An ELISA assay using an anti-(β-GGLs) monoclonal antibody indicated that the synthetic homologues with long acyl chains had greater diagnostic potential in the order C18 > C16 > C14 > C12. Toward a simultaneous detection of natural glycolipids by mass spectrometry (MS), a deuterium-labeled C16 homologue (β-GGL-C16-d3) was prepared and applied as an internal standard for a high-resolution electrospray ionization MS (ESI-MS) analysis. The ESI-MS analysis was used to identify and quantify acyl homologues (C16/C16, C16/C18, and C18/C18) of β-GGL-C16 in cultured M. pneumoniae. A β-GGLs homologue with a 1,2-diacetyl group (C2) was also prepared as a "water soluble" glycolipid homologue and characterized by 1H NMR spectroscopy. We envisage that each of these chemosynthetic homologues will provide promising approaches to solve medical and biological problems associated with mycoplasma infectious diseases (MIDs).