The aim of investigation was to find the differences in the content and composition of essential oil between 16 ecotypes of St. John’s wort growing wild in the different localities in South-Eastern Poland. The herb was collected during full blooming. Significant differences both in the content and composition of essential oil of investigated populations were found. The essential oil content ranged from 0.10% to 0.47%. Its major constituents were 2-methyloctane and α-terpineol.