Chemical composition and essential oil yields from aerial parts of 31 Iranian Achillea millefolium accessions, each collected from their natural habitats of Iran and grown together in field conditions, were investigated. The concentrations of the hydro-distilled essential oils ranged from 0.03% to 0.39%. Gas chromatography–mass spectrometry (GC–MS) analysis revealed 50 compounds in the accessions. The main components of the essential oils in Iranian A. millefolium accessions varied in the following ranges: 1,8-Cineole, 1.2–19.8%; β-thujone, 0.4–55.3%; camphor, 0.6–25.5%; germacrene-D, 2–20.6%; trans-nerolidol, 0.4–48.1%; isospathulenol, 0.5–36%; and cubenol, 0.1–42.9%. According to cluster analysis, five chemotypes were obtained as 1,8-Cineole/trans-nerolidol, high cubenol, high germacrene-D/isospathulenol, high camphor/cubenol, and high 1,8-Cineole/ β-thujone/cubenol. The result of principal component analysis (PCA) indicated that germacrene-D and isospathulenol components were under more genetic control than the other main components. Results revealed a high level of variation of composition and yield of essential oils among the Iranian A. millefolium accessions.