Phytosterols in commercially grown Runner, Virginia, and Spanish peanuts (n = 221) from 2005 and 2006 were quantified by a combination of acid hydrolysis and alkaline saponification steps followed by gas chromatography-flame ionization detection and gas chromatography-mass spectrometry analysis of the trimethylsilyl derivatives. Δ(5)-Avenasterol, which partially degrades during acid hydrolysis, was quantified after alkaline saponification plus direct analysis of the steryl glucosides isolated by solid-phase extraction. β-Sitosterol, Δ(5)-avenasterol, campesterol, and stigmasterol were identified in peanut lipid extracts as the dominant sterols by retention time mapping and mass spectra with recoveries ∼99%. Clerosterol, Δ(5,24(25))-stigmastadienol, Δ(7)-sitosterol + cycloartenol, and one unidentified sterol were also present but at low levels. Free and esterified phytosterols accounted for ∼80% of the total sterols determined; the remainder was attributed to steryl glucosides. The total sterol level in Spanish market type peanuts (144.1 ± 5.3 mg/100 g) was significantly greater than both Runners (127.5 ± 6.3 mg/100 g) and Virginias (129.3 ± 6.9 mg/100 g) (P < 0.05). Tamspan 90 (146.9 mg/100 g) followed by OLIN (138.5 mg/100 g) showed the highest total sterol content among the cultivars examined. Cultivar effects were strongly significant (P < 0.001) for all phytosterols, whereas production year effects were strongly significant (P < 0.001) for Δ(5)-avenasterol, Δ(5,24(25))-stigmastadienol, and the combined quantities of Δ(7)-sitosterol + cycloartenol, which coeluted. Cultivar × year interactions were strongly significant (P < 0.001) in all sterols except for Δ(7)-sitosterol + cycloartenol (P < 0.01). Total phytosterol contents were markedly higher than those reported in the existing literature for Runner and Virginia type peanuts, partially attributed to the inclusion of steryl glucosides in the analysis.