In this work, fast and sensitive high-performance liquid chromatography (HPLC) coupled with multivariate analysis was utilized to evaluate the metabolic profiling of Jatropha curcas seed and screen the marker compounds of phorbolesters (PEs), which significantly contributed to the metabolic profiling for quality control of PEs in J. curcas seed. Thirty-two peaks were separated and detected in J. curcas seed on a fused-core C(18) column between 2 and 25 min. Principal component analysis (PCA) and hierarchical cluster analysis (HCA) of the chromatographic data demonstrated that 12 batches of J. curcas seed could be well-differentiated and categorized into 5 groups. Especially, one sample obtained from Lijiang Yunan was significantly different from the others. Partial least-squares discriminant analysis (PLS-DA) showed that the metabolic profiling differentiation might be explained by 9 components including J.factors C(1)-C(5) of PEs, peak 21 (similar to J.factor C(1)) and 3 peaks (peaks 1, 9, and 10) significantly different in retention time from the PEs. The observation that the content levels of J.factors C(1) and C(2) plus peak 21 (equivalent to J.factor C(1)) were more relevant to the multivariate chromatographic data than the ones of J.factors C(3)-C(5) was confirmed by the PLS prediction models. The results of the present study indicated not only that J.factors C(1) and C(2) were the more rational markers representing the comprehensive quality of PEs in J. curcas seed but also that peak 21 (similar to J.factor C(1)) was a rational marker, too.