Processing of hoki, a commercially important fish species, generates substantial quantities of co-products, including male gonad, which contains valuable lipids, such as phospholipids, that could be recovered and utilised. Hoki fish male gonads (HMG) were subjected to pulsed electric fields (PEF) treatment at varying field strengths (0.625, 1.25, and 1.875 kV/cm) and frequencies (25, 50, and 100 Hz), at a fixed pulse width of 20 μs. The total lipid was extracted using an ethanol-hexane-based (ETHEX) extraction method, and the phospholipid and fatty acid compositions were determined using 31P NMR and GC-FID, respectively. The total lipid yield was increased from 4.1% to 6.7% by a relatively mild PEF pre-treatment at a field strength of 1.25 kV/cm and frequency of 50 Hz. A higher amount of EPA (8.2%), DPA (2.7%), and DHA (35.7%) were obtained by that treatment, compared to both un-heated (EPA: 8%; DPA: 2.5%; DHA: 35.2%) and heat-treated controls (EPA: 7.9%; DPA: 2.5%; DHA: 34%). No significant changes to the content of the major phospholipids were observed. PEF pre-treatment under mild conditions has potential for improving the total lipid yield extracted from fish male gonad.