Abstract: Isoprenoidal glycerol dibiphytanyl glycerol tetraethers (iso-GDGTs) and branched glycerol dialkyl glycerol tetraethers (br-GDGTs) have been widely used as biomarkers of archaea and bacteria for the investigation of paleoenvironment. The analysis of GDGTs was generally carried out by separation using high performance liquid chromatography (HPLC), followed by mass spectrometry in the selected ion monitoring mode with soft ionization techniques such as atmospheric chemical ionization (APCI). However, previous studies have shown that approximate mass selection for GDGT analysis using selected ion monitoring generates losses of relative GDGT signals of up to 36%, which will make it difficult to determine the paleoenvironmental parameters based on the distribution of GDGTs accurately. Furthermore, the traditional method is only limited to the targeted detection of reported GDGTs which have been used in paleoenvironmental parameters, could not carry out a comprehensive analysis of the samples, leading to the loss of potential GDGT homologues. Here, an approach was described for the characterization of GDGTs by high resolution mass spectrometry, which could perform non-targeted detection of GDGTs, and identify target ions with structural information through tandem mass spectrometry. A total of 14 GDGTs, including 6 iso-GDGTs with 0 to 6 alicyclic rings and 8 br-GDGTs with different alkyl side chains, were assigned by accurate mass values from the mass spectra of surface sediments from the Pearl River Estuary. The analysis of tandem mass spectrometry showed that GDGTs have multiple fragmentation pathways, GDGT-0, crenarchaeol and br-GDGT Ia all have at least 7 fragmentation pathways. The fragment ions were divided into oxygen-containing fragment ions and hydrocarbon fragment ions, which correspond to the characteristic fragmentation pathways of its precursor ions and the alkyl chains, respectively. Fragment ions of hydrocarbon moities were used as the fingerprint, which were combined with oxygen-containing fragment ions to describe the fragmentation behavior of precursor ions. The ultra-high mass resolution provided by Orbitrap MS can meet the direct analysis of GDGTs, and it is a successful tool to study the molecular composition of GDGTs from complex matrices. In addition, the key advantage of APPI-P Orbitrap MS is the ability to detect species not part of classical targeted GDGTs. Unfortunately, the method is not coupled with liquid chromatography, and the isomers of GDGT cannot be detected. This requires future efforts. The results showed great potential for the use of APPI-P Orbitrap MS as both a rapid method for determining novel GDGTs and perhaps more significantly, as a tool for the rapid detection of putative homologues that may establish novel proxies between the paleoenvironmental variables and distribution of GDGTs.