Fennel (Foeniculum vulgare Mill.) is an old medicinal plant and has been commonly used as a traditional food and medicine. This study aimed to evaluate genetic variation for agro-morphological traits and essential oil content and components in a worldwide collection of bitter fennel (F. vulgare var. vulgare). We examined production stability of different accessions, and identified associations between traits in order to find the best criteria for use in future breeding programs. The study evaluated 33 bitter fennel accessions from 11 countries in the field for morphological characters across 4 years (2015-18), and for important phytochemical characters in 2 years (2015 and 2016). Comparisons across years revealed varied responses from fennel genotypes. Highest mean production was observed in the third year, and highest essential oil content in the second year. Based on gas chromatography-mass spectrometry analysis, trans-anethole (59.86-78.82% of total oil content), estragole (3.61-24.39%), fenchone (9.58-18.26%) and limonene (0.52-3.66%) were detected as major essential oil components in the studied germplasm. Based on principal component and cluster analysis, genotypes were grouped into different clusters. Classification of genotypes was mainly in agreement with available information regarding their origins. The results support available information on the history of human establishments and the introduction of fennel from Mediterranean gene pools to other regions in ancient times. The negative correlation between some essential oil components indicates that some components are isomers and this could limit the simultaneous selection of components. Superior genotypes were identified in the studied germplasm, which can be used for future breeding programs with specific purposes.