Seed oil percentage (SOP) and fatty acid composition of watermelon (Citrullus lanatus) seeds are important traits in Africa, the Middle East, and Asia where the seeds provide a significant source of nutrition and income. Oil yield from watermelon seed exceeds 50% (w/w) and is high in unsaturated fatty acids, a profile comparable to that of sunflower (Helianthus annuus) and soybean (Glycine max) oil. As a result of novel non-food uses of plant-derived oils, there is an increasing need for more sources of vegetable oil. To improve the nutritive value of watermelon seed and position watermelon as a potential oil crop, it is critical to understand the genetic factors associated with SOP and fatty acid composition. Although the fatty acid composition of watermelon seed is well documented, the underlying genetic basis has not yet been studied. Therefore, the current study aimed to elucidate the quality of watermelon seed oil and identify genomic regions and candidate genes associated with fatty acid composition. Seed from an F2 population developed from a cross between an egusi type (PI 560023), known for its high SOP, and Strain II (PI 279261) was phenotyped for palmitic acid (16:0), stearic acid (18:0), oleic acid (18:1), and linoleic acid (18:2). Significant (P < 0.05) correlations were found between palmitic and oleic acid (0.24), palmitic and linoleic acid (–0.37), stearic and linoleic acid (–0.21), and oleic and linoleic acid (–0.92). A total of eight quantitative trait loci (QTL) were associated with fatty acid composition with a QTL for oleic and linoleic acid colocalizing on chromosome (Chr) 6. Eighty genes involved in fatty biosynthesis including those modulating the ratio of saturated and unsaturated fatty acids were identified from the functionally annotated genes on the watermelon draft genome. Several fatty acid biosynthesis genes were found within and in close proximity to the QTL identified in this study. A gene (Cla013264) homolog to fatty acid elongase (FAE) was found within the 1.5-likelihood-odds (LOD) interval of the QTL for palmitic acid (R2 = 7.6%) on Chr 2, whereas Cla008157, a homolog to omega-3-fatty acid desaturase and Cla008263, a homolog to FAE, were identified within the 1.5-LOD interval of the QTL for palmitic acid (R2 = 24.7%) on Chr 3. In addition, the QTL for palmitic acid on Chr 3 was located ≈0.60 Mbp from Cla002633, a gene homolog to fatty acyl- [acyl carrier protein (ACP)] thioesterase B. A gene (Cla009335) homolog to ACP was found within the flanking markers of the QTL for oleic acid (R2 = 17.9%) and linoleic acid (R2 = 21.5%) on Chr 6, whereas Cla010780, a gene homolog to acyl-ACP desaturase was located within the QTL for stearic acid (R2 = 10.2%) on Chr 7. On Chr 8, another gene (Cla013862) homolog to acyl-ACP desaturase was found within the 1.5-LOD interval of the QTL for oleic acid (R2 = 13.5%). The genes identified in this study are possible candidates for the development of functional markers for application in marker-assisted selection for fatty acid composition in watermelon seed. To the best of our knowledge, this is the first study that aimed to elucidate genetic control of the fatty acid composition of watermelon seed.