Oil content and fatty acid composition profiles in grain of 18 proso millet accessions were investigated. The results showed that oil content in proso millet ranged from 2.54% to 4.00%, and averaged 3.71%. The major fatty acids detected were linoleic (18:2n6), oleic (18:1n9) and palmitic (16:0), ranging from 0.075 to 5.19, 0.150 to 3.02, and 0.897 to 1.81 g/100 g, respectively. Minor fatty acids included palmitoleic, stearic, linolenic, arachidic, arachidonic, docosanoic and tetracosanoic acids. The unsaturated fatty acids (UFA) accounted for 82.1%~94.6% of the total oil. Both correlation analysis and principal component analysis indicated significant inter-relationships among ten fatty acids. Cluster analysis suggested that the fatty acid profile was significantly related to genotypes.