Volatile thiol 3-mercaptohexan-1-ol (3MH) and particularly 4-mercapto-4-methylpentan-2-one (4MMP) are highly potent flavour compounds in hops. For the determination, a simple and robust stable isotope dilution LC–MS/MS method was developed and applied to 32 hop varieties worldwide from harvest years 2019 and 2020. Limit of detection, precision, and recovery were 0.15 μg/kg, 10%, and 97–108%, respectively. Levels of 3MH and 4MMP ranged from 1.9 to 79.2 μg/kg and from undetectable to 37.1 μg/kg, respectively. Citra, Mosaic, and Strata were rich in both thiols. ICP analyses revealed, that variation of potassium content between the two harvest years was inversely correlated with that of manganese and rubidium (|r| ≥ 0.89) among 12 US varieties excluding Citra and Mosaic. Total essential oil content (0.34–2.7 mL/100 g) was inversely correlated with calcium content (|r| ≥ 0.65). Greatly varying thiol levels depending on variety, region and harvest year might lead to differing flavour results in beer. •A simple and robust LC–MS/MS method for free thiol analysis in hops was established.•US Citra, Mosaic and Strata were rich in both thiols.•Ca content was inversely correlated with total essential oils (Pearson's |r| ≥ 0.65).•Remarkable deviation in K & P contents (14%) was seen in Mosaic between two harvests.