Emerging magnetic resonance imaging (MRI) biomarkers of hepatic steatosis have demonstrated tremendous promise for accurate quantification of hepatic triglyceride concentration. These methods quantify the proton density fat‐fraction (PDFF), which reflects the concentration of triglycerides in tissue. Previous in vivo studies have compared MRI‐PDFF with histologic steatosis grading for assessment of hepatic steatosis. However, the correlation of MRI‐PDFF with the underlying hepatic triglyceride content remained unknown. The aim of this ex vivo study was to validate the accuracy of MRI‐PDFF as an imaging biomarker of hepatic steatosis. Using ex vivo human livers, we compared MRI‐PDFF with magnetic resonance spectroscopy‐PDFF (MRS‐PDFF), biochemical triglyceride extraction, and histology as three independent reference standards. A secondary aim was to compare the precision of MRI‐PDFF relative to biopsy for the quantification of hepatic steatosis. MRI‐PDFF was prospectively performed at 1.5 Tesla in 13 explanted human livers. We performed colocalized paired evaluation of liver fat content in all nine Couinaud segments using single‐voxel MRS‐PDFF (n = 117) and tissue wedges for biochemical triglyceride extraction (n = 117), and five core biopsies performed in each segment for histologic grading (n = 585). Accuracy of MRI‐PDFF was assessed through linear regression with MRS‐PDFF, triglyceride extraction, and histology. Intraobserver agreement, interobserver agreement, and repeatability of MRI‐PDFF and histologic grading were assessed through Bland‐Altman analyses. MRI‐PDFF showed an excellent correlation with MRS‐PDFF (r = 0.984, confidence interval 0.978‐0.989) and strong correlation with histology (r = 0.850, confidence interval 0.791‐0.894) and triglyceride extraction (r = 0.871, confidence interval 0.818‐0.909). Intraobserver agreement, interobserver agreement, and repeatability showed a significantly smaller variance for MRI‐PDFF than for histologic steatosis grading (all P < 0.001). Conclusion: MRI‐PDFF is an accurate, precise, and reader‐independent noninvasive imaging biomarker of liver triglyceride content, capable of steatosis quantification over the entire liver. (Hepatology 2015;62:1444–1455)