Oil content, fatty acids profile, acid and saponification values of poppy seeds grown on two localities of the Slovak Republic were evaluated in the study. Statistically significant effects of locality, genotype and their interaction (P < 0.05) for numerous descriptors were proved by non-parametric tests. Results confirmed that variation in the analysed parameters was influenced by the colour of seeds. Ochre variety Redy contained the highest oil level in both localities (49.9 and 52.4%) and linoleic acid level (74.3 and 71.6%). White-seeded Racek and Albín had the highest acid value (2.8 and 2.4% of free fatty acids) and grey-seeded Malsar and blue-seeded Maratón contained the highest saponification value. Buddha, a high-morphine poppy variety, differed significantly in all monitored parameters. High negative interrelation between linoleic and oleic acids levels was observed. Oil content was positively correlated with linoleic acid and negatively with oleic acid. Weather conditions at the end of vegetation influenced the accumulation of oil and essential linoleic acid.