First-generation SARS CoV-2 vaccines that generate immune responses to ancestral Spike glycoprotein sequences have averted at least 14.4 million deaths, but their effectiveness against the recently emerged Omicron lineages is reduced. The updating of booster vaccines with variant Spike sequences is therefore likely required to maintain immunity as the pandemic continues to evolve. The Spike is a trimeric integral membrane protein with a membrane spanning sequence at its C-terminus. The Spike protein-based vaccine that is currently licensed for human use is produced by a complex process that reconstitutes the Spike in an artificial membrane. Alternatively, production of the Spike trimer as a soluble protein generally requires replacement of the membrane spanning sequence with a foreign often highly immunogenic trimerization motif that can complicate clinical advancement. We used systematic structure-directed mutagenesis coupled with functional studies to identify an alternative stabilization approach that negates the requirement for an external trimerization motif or membrane-spanning sequence. The replacement of 2 alanine residues that form a cavity in the core of the Spike trimer with bulkier hydrophobic residues resulted in increased Spike thermal stability. Thermostable Spike mutants retained major conserved neutralizing antibody epitopes and the ability to elicit broad and potent neutralizing antibody responses. One such mutation, referred to as VI, enabled the production of intrinsically stable Omicron variant Spike ectodomain trimers in the absence of an external trimerization motif. The VI mutation potentially enables a simplified method for producing a stable trimeric S ectodomain glycoprotein vaccine.