The growing awareness of a mounting trend toward adulteration of essential oil products has resulted in considerable interest among producer and consumer industries to develop suitable and cost-effective methods for authenticity control. In response to this trend, this study reports authenticity assessment of Australian tea tree oil (TTO) by means of stereospecific analysis of designated chiral monoterpenes present within the oil. Enantioselective gas chromatography (. eGC) coupled with either flame ionization detection or mass spectrometry was used to determine the enantiomeric distribution of terpinen-4-ol and a-terpineol, both key chemical markers in TTO. Consistent enantiomeric ratios of 68.5. ?. 0.2 (+): 31.5. ?. 0.2 (-) for terpinen-4-ol and 74.2. ?. 1.4 (+): 25.8. ?. 1.4 (-) for a-terpineol were observed for the 57 authentic 100 pure Australian TTOs analyzed, which were sourced directly from different plantation sites throughout Australia. On the other hand, considerable variations from these ratios were observed for a number of commercial TTOs sampled from different continents, with 50 of the samples varying markedly from the 68.5 value found for (+) terpinen-4-ol. Robustness of the method and reliability of the attained results were evaluated by cross-checking data derived from the two independent analyses performed in this study. To the best of our knowledge, this work encapsulates the most comprehensive dataset currently available for the enantiomeric distribution of terpinen-4-ol and a-terpineol in pure Australian TTO, and which can serve as an important reference for detection of variation in commercially-sourced samples, and be a potential indicator of adulteration. Additionally, results of this study proved the considerable potential of using chiral compositions of terpinen-4-ol and a-terpineol as chiral signatures for authenticity assessment of TTO.