This study compared the effects of hemp seed oil versus four different UFA-rich seed oils in the diet of laying hens on egg production, egg quality, and fatty acid profile of the yolk. Soybean oil (SBO), sunflower oil (SFO), corn oil (CO), canola oil (CAO), and hemp seed oil (HSO) were included in the hens’ diets in equal proportions. A total of one hundred and twenty White Leghorn hens were allocated into five groups with 8 replicates, each with 3 hens. The trial lasted 84 days and data were collected on egg production, quality, and fatty acid profile of the yolk. The results showed that none of the incorporated seed oils affected egg production parameters and eggshell quality. However, hemp seed oil altered yolk colour values similarly to canola oil by increasing the L* value of the yolk whilst decreasing the a* value (P < 0.05). Hemp oil increased the PUFA content in the yolk, similar to soybean, corn, and sunflower oil, but unlike the latter, it also enriched the n-3 fatty acids in the yolk (P < 0.05). In conclusion, hemp seed oil can be safely used in the diet of chickens without negative effects on egg production and egg quality like other seed oils. Furthermore, hemp seed oil can improve the desirable fatty acid content in the yolk and has the potential to produce n-3-enriched eggs.