SummaryHop (Humulus lupulus L.) inflorescence, commonly known as ‘hop cone’, is valuable for contents of bitter acids, essential oils and polyphenols, used in brewing industry and biomedical application. These compounds are predominantly formed in glandular trichomes, known as ‘lupulin glands’. In this study, we investigated chemical and morphological characteristics of lupulin glands by microscopy and HPLC analyses. Even though no significant correlations were found between lupulin glands and hop cones for contents of alpha and beta bitter acids, polyphenols, all ratios between individual metabolites (alpha/beta acids, X/alpha acids, X/DMX) were highly and significantly correlated. The average number of lupulin glands on bract/bracteole ranged from 115 to 713 with 28.5% of variability. Linear regression analyses confirmed that total volume of lupulin glands and total lupulin glands area on bract/bracteole surface highly correlated with contents of bitter acids and polyphenols in hop cone. Therefore, lupulin glands are unique and exclusive organs responsible for contents of bitter acids and polyphenols in hop cone.