Wort from the barley varieties (Hordeum vulgare) Pallas, Fero, and Archer grown on the same location were investigated for their influence on oxidative stability and volatile profile during wort processing. Barley varieties had a small influence on radical formation, thiol-removing capacity, and volatile profile. Wort boiling with and without hops had a large influence on these same parameters. Potentially antioxidative thiols were oxidized in sweet wort, but reduction of thiols using tris(2-carboxyethyl)phosphine hydrochloride revealed that Archer wort had a significantly larger content of total thiols than Pallas and Fero. Oxidized thiols resulted in gel proteins and longer filtration time for Archer wort. Our study shows that wort processing to a large extent will eliminate variations in volatile profile and thiol levels in wort which otherwise might arise from different barley varieties.