Technical Abstract: Castor has multiple industrial applications including potential as a feedstock for biodiesel production. The oil content and fatty acid composition in castor seed are important factors to determine the price for production and affect the key fuel properties of biodiesel. The entire U.S. castor germplasm collection (1033 available) was screened for oil content and fatty acid composition by nuclear magnetic resonance (NMR) and gas chromatography (GC), respectively. Castor seeds on the average contain 48.2% oil with a significant variability ranging from 37.2 – 60.6%. Methyl esters were prepared from castor seed by alkaline transmethylation. GC analysis of methyl esters confirmed that castor oil was comprised primarily of eight fatty acids: 1.48% palmitic (C16:0), 1.58% stearic (C18:0), 4.41% oleic (C18:1), 6.42% linoleic (C18:2), 0.68% linolenic (C18:3), 0.45% gadoleic (C20:1), 84.51% ricinoleic (C18:1-1OH), and 0.47% dihydroxystearic (C18:0-2OH) acids. Significant variability on fatty acid composition was detected among castor accessions. Ricinoleic acid (RA) was positively correlated with dihydroxystearic acid (DHSA) but highly negatively correlated with the other five fatty acids except linolenic acid. Based on oil content and fatty acid profile, 668 accessions were selected for genotyping with 26 SSR markers. Five clusters were identified from genetic analysis and genetic diversity existed in the collection. According to the genetic analysis results, 206 accessions will be selected for morphological observation in the field to help establishing the U.S. castor core collection. The results obtained from chemical and genetic analyses will be useful for end-users to explore castor germplasm for biodiesel production.