Six species of Balistoidei (Stephanolepis cirrhifer, Canthidermis maculata, Triacanthus biaculeatus, Monacanthus chinensis, Thamnaconus hypargyreus, and Aluterus monoceros) were collected from the market of Zhanjiang City, Guangdong Province of China to compare the fatty acid composition in their muscles. The moisture and crude fat contents were measured using the Soxhlet extraction method according to national standard. The determination of fatty acid components in the muscle samples of Balistoidei was performed using capillary gas chromatography, and 24 fatty acid components were identified. The contents of saturated fatty acid (SFA), monounsaturated fatty acid (MUFA), and polyunsaturated fatty acid (PUFA) were analyzed and compared respectively. The results showed that the moisture content in Balistoidei body ranged from 67.21% to 72.95%, and the crude fat content ranged from 1.05% to 1.42% of dry weight. The contents of SFA, MUFA, and PUFA in muscles of the six Balistoidei species were in the range of 34.21%–43.45%, 11.84%–19.43%, and 34.39%–48.86%, respectively. The content of PUFA was relatively higher than that of SFA and MUFA. ω-3 fatty acid content was higher than ω-6 fatty acid content. The ω-3/ω-6 ratios ranged from 1.0 to 4.7, indicating a relatively high fatty acid nutritional value. In addition, the ω-3/ω-6 ratios of C. maculata and T. hypargyreus were 3.3 and 4.7, respectively. The content of eicosapentaenoic acid (EPA) + docosahexaenoic acid (DHA) in the muscles was high (17.20%–37.49%) in all the six species and in the order of T. hypargyreus (37.49%) > C. maculata (30.29%) > M. chinensis (25.73%) > S. cirrhifer (23.82%) > T. biaculeatus (18.82%) > A. monoceros (17.20%). The EPA + DHA content in T. hypargyreus was as high as 37.49%±2.67%, indicating high edible and medicinal values.