This study has utilized the tools of lipid biomarker chemistry and molecular phylogenetic analyses to assess the archaeal contribution to diversity and abundance within a microbial mat and underlying sediment from a hypersaline lagoon in Baja California. Based on abundance of ether-linked isoprenoids, archaea made up from 1 to 4% of the cell numbers throughout the upper 100 mm of mat and sediment core. Below this depth archaeal lipid was two times more abundant than bacterial. Archaeol was the primary archaeal lipid in all layers. Relatively small amounts of caldarchaeol (dibiphytanyl glyceroltetraether) were present at most depths with phytanyl to biphytanyl molar ratios lowest (~10 : 1) in the 4–17 mm and 100–130 mm horizons, and highest (132 : 1) in the surface 0–2 mm. Lipids with cyclic biphytanyl cores were only detected below 100 mm. A novel polar lipid containing a C30 isoprenoid (squalane) moiety was isolated from the upper anoxic portion of the core and partially characterized. Hydrocarbon biomarker lipids included pentamethylicosane (2–10 mm) and crocetane (primarily below 10 mm). Archaeal molecular diversity varied somewhat with depth. With the exception of samples at 0–2 mm and 35–65 mm, Thermoplasmatales of marine benthic group D dominated clone libraries. A significant number of phylotypes representing the Crenarchaeota from marine benthic group B were generally present below 17 mm and dominated the 35–65 mm sample. Halobacteriaceae family made up 80% of the clone library of the surface 2 mm, and consisted primarily of sequences affiliated with the haloalkaliphilic Natronomonas pharaonis.