The Hibiscus genus encompasses more than 300 species, but kenaf (Hibiscus cannabinus L.) and roselle (Hibiscus sabdariffa L.) are the two most economically important species within the genus. Seeds from these two Hibiscus species contain a relatively high amount of oil with two unusual fatty acids: dihydrosterculic and vernolic acids. The fatty acid composition in the oil can directly affect oil quality and its utilization. However, the variability in oil content and fatty acid composition for these two species is unclear. For these two species, 329 available accessions were acquired from the USDA germplasm collection. Their oil content and fatty acid composition were determined by nuclear magnetic resonance (NMR) and gas chromatography (GC), respectively. Using NMR and GC analyses, we found that Hibiscus seeds on average contained 18% oil and seed oil was composed of six major fatty acids (each >1%) and seven minor fatty acids (each <1%). Hibiscus cannabinus seeds contained significantly higher amounts of oil (18.14%), palmitic (20.75%), oleic (28.91%), vernolic acids (VA, 4.16%), and significantly lower amounts of stearic (3.96%), linoleic (39.49%), and dihydrosterculic acids (DHSA, 1.08%) than H. sabdariffa seeds (17.35%, 18.52%, 25.16%, 3.52%, 4.31%, 44.72%, and 1.57%, respectively). For edible oils, a higher oleic/linoleic (O/L) ratio and lower level of DHSA are preferred, and for industrial oils a high level of VA is preferred. Our results indicate that seeds from H. cannabinus may be of higher quality than H. sabdariffa seeds for these reasons. Significant variability in oil content and major fatty acids was also detected within both species. The variability in oil content and fatty acid composition revealed from this study will be useful for exploring seed utilization and developing new cultivars in these Hibiscus species.