The presence and role of polysulfide radicals in the electrochemical processes of lithium sulfur (Li–S) batteries is currently being debated. Here, first-principles interpretations of measured X-ray absorption spectra (XAS) of Li–S cells are leveraged with an ether-based electrolyte. Unambiguous evidence is found for significant quantities of polysulfide radical species (LiS3, LiS4, and LiS5), including the trisulfur radical anion S3 −, present after initial discharge to the first discharge plateau, as evidenced by a low energy shoulder in the S K-edge XAS below 2469 eV. This feature is not present in the XAS of cells at increased depth of discharge, which, by our analysis, exhibit increasing concentrations of progressively shorter polysulfide dianions. Through a combination of first-principles molecular dynamics and associated interpretation of in situ XAS of Li–S cells, atomic level insights into the chemistries are provided that underlie the operation and stability of these batteries.