Garlic (Allium sativum), belonging to the family Alliaceae, is a widely distributed plant used throughout the world not only as a spice and a food, but also as a folk-medicine. In this study, the bulbs of various populations of the plant were collected from different areas in Iran. The total alliin, allicin and alliinase contents of the garlic ecotypes were determined by HPLC system. The data were subjected to analysis of variance based on the model of completely randomized design with three replications. A significant difference (P<0.01) in alliin, allicin and alliinase content was obtained from the different ecotypes. Results indicated that the highest mean of alliin content (27.36%) was obtained in Heydareh (Hamadan) province, the highest mean of allicin content (25.66%) in Tu'in (Hamadan) province and the highest mean of alliinase content (27.33%) was observed in Sabzevar province. Since morphological characteristics can vary under different agroclimatic conditions, interactions between genotype and environment can complicate the characterization of garlic clones. In conclusion, Heydareh, Tu'in and Sabzevar as the superior garlic ecotypes have the best genetic potential to use in the breeding programmes.