Physicochemical and instrumental characterization of rubber (Hevea brasiliensis Müll. Arg.) seed oil (RSO) was carried out for the purposes of quality assessment, identification, and authentication. Properties such as color (Lovibond), specific gravity, percent FFA (as oleic acid), acid value, saponification value, iodine value, and viscosity were determined. FA composition and M.W. averages of RSO were determined using GLC and gel permeation chromatography (GPC), respectively. Structural features of RSO were also determined using FTIR, 1 H NMR, and 13 C NMR spectroscopy. The natural form of RSO is highly acidic (acid value ≈ 43.6 mg KOH/g). The saturated FA are palmitic (17.50%) and stearic (4.82%), and the main unsaturated FA are oleic (25.33%), linoleic (37.50%), and linolenic (14.21%). The oil can be classified as semidrying. GPC shows an unusual peak that is due to a very high M.W. (≈38,800) fraction that is not found in the chromatogram of known vegetable oils and is therefore unique to RSO. FTIR, 1 H NMR, and 13 C NMR analyses confirmed that RSO is composed mainly of TAG of saturated and unsaturated FA. Functional groups such as carbonyl, olefinic unsaturation, esters, glyceryl, methylene, and terminal methyl that are present in vegetable oils are also present in RSO.