Omega-3 long-chain polyunsaturated fatty acids (ω-3 LC-PUFAs), particularly eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), are obtained from oleaginous microalgae and fish. These LC-PUFAs are vital for human health, contributing to cardiovascular health, brain function, and fetal development. Their intake also reduces the risk of chronic diseases such as heart disease, miscarriages, mental disorders, etc. Traditionally, marine fish and microalgae have been the primary sources of LC-PUFAs, but their diminishing natural populations and limitations in their cultivation have led to the shift of focus on oil from oilseeds and yeasts. Oilseeds and yeasts transformed with elongases and desaturases from fish with high substrate specificity toward ω-3 fatty acids can be used to produce oils with EPA and DHA. Likewise, transgenic yeast producing EPA and DHA can overcome the low productivity of microalgae and provide a cheaper alternative to oils from microbes. Moreover, genes involved in rate-limiting steps can be regulated and modified to overcome limitations like substrate dichotomy and low oil accumulation. Thus, pathway engineering in oilseeds and yeasts could pave the way for sustainable, commercial-scale production of EPA and DHA in genetically modified organisms promising ω-3-rich oils, meeting global health and nutritional demands.