Over the last decade, glycerol dialkyl glycerol tetraethers (GDGTs) have become one of the most investigated lipid classes in marine and terrestrial organic geochemical research. GDGTs are microbial membrane core lipids biosynthesized as multiple homolog series of isoprenoid or methyl-branched isomers [isoprenoid glycerol dialkyl glycerol tetraethers (isoGDGTs) and Branched GDGTs (brGDGTs), respectively], whose relative abundance depend on a range of environmental parameters, including temperature. This has led to the development of GDGT-based temperature proxies. A key aspect in the analysis of GDGTs and the further development of their use as environmental proxies is good chromatographic separation of the full range of structural and stereo-isomers, with potential for discovery of novel GDGT variants. Several HPLC methods have been developed to this extent, but partial co-elution of GDGTs remains an issue despite long run times. In this study, we investigate the effects of different types of reverse phase (RP) chromatography on the separation of GDGT isomers. We found that the use of a Kinetex C18-XB column gives good separation of isoGDGT isomers in comparison to the recently developed double column HILIC analysis operated in normal phase (NP) and has a shorter run time. In marine samples, the regularly reported isoprenoid GDGTs separated in a similar way as in NP, however an earlier eluting group was observed to elute with the crenarchaeol isomer used in the TEX₈₆ proxy. In a Swedish peat bog sample, a large range of isoGDGT isomers were observed. We observed a range of brGDGT isomers in several samples often with near baseline separation. Exact identification of all these isomers remained elusive, due to the different mechanism of separation in RP, and the complexity of the brGDGT family. The C18-XB method is rapid and versatile and can be set up on either low-pressure HPLC systems (max 400 bar) with a sample run time of 25 min for brGDGTs and 45 min to include isoGDGTs. On UHPLC-MS systems (>600 bar) the sample run time is reduced to 15 min. Most importantly, the C18-XB method presented here gives unusual separation of both isoprenoid and brGDGTs and could be a useful tool for the further elucidation of the biological sources and environmental factors that play a role in the production of different GDGT isomers.