Tree Wormwood (Artemisia arborescens L.) is a Mediterranean medicinal herb known for its anti-inflammatory properties, mainly because of the high chamazulene content of its essential oil (EO). Herein are presented the results of an investigation concerning the seasonal variation in the chamazulene content of.the EOs of three Cretan natural populations of A. arborescens by combined GC-FID and GC-MS analysis. Results highlighted a population containing EO rich in chamazulene at amounts exceeding 50% of its quantitative composition at the full flowering stage, which is the more appropriate vegetative stage for harvesting. Hydro-distillation of the respective plant material in five different pH environments increased, in all cases, the chamazulene yield, with the most efficient pH value being 8.3, when the chamazulene amount increased 1.3 times compared with distillation under neutral conditions, reaching 65.1% of the total content of EO.