Medicinal plants were considered as valuable genetic germplasms in Iran. One of these medicinal plants is fennel. Fennel ( Foeniculum vulgare Mill) belongs to Apiaceae family. It is a perennial medicinal and spice plant. Fennel is cultivated in different regions of Iran. The aims of the present study were to evaluate the inbreeding effect and harvesting time on secondary metabolites and to assess the genetic variation among self and outcross pollinated fennel ecotypes using morphological, anatomical and molecular markers. Furthermore, the essential oil compositions of different seed ecotypes were evaluated using GC-MS analysis. This study was performed using factorial experiment in Randomized Complete Block Design (RCBD) in three replicates in 1391-1393. In this research, 15 ISSR primers were used in which 248 were polymorphic. The polymorphism percent was 82.33 and the PIC was 0.42. The ISSR primers 0in 0in 0pt" Keywords : Inbreeding, Fennel, Trans -anethole, Polyphenols