Pruni Semen, the seed of several unique Prunus plants, is a traditional purgative herbal material. To determine the authentic sources of Pruni Semen, 46 samples from four species were collected and analyzed. Ten compounds including multiflorin A (Mul A), a notable purative compound, were isolated and identified by chemical separation and nuclear magnetic resonance spectroscopy. Seventy-six communal components were identified by ultra-high performance liquid chromatography with linear ion trap-quadrupole Orbitrap mass spectrometry, and acetyl flavonoid glycosides were recognized as characteristic constituents. The flavonoids were distributed in the seed coat and cyanogenic glycosides in the kernel. Based on this, methods for identifying Pruni Semen from different sources were established using chemical fingerprinting, quantitative analysis of the eight principal compounds, hierarchical cluster analysis, principal component analysis, and orthogonal partial least squares discriminant analysis. The results showed that the samples were divided into two categories: one is the small seeds from Prunus humilis (Ph) and Prunus japonica (Pj), and the other is the big seeds from Prunus pedunculata (Pp) and Prunus triloba (Pt). The average content of Mul A was 3.02, 6.93, 0.40, and 0.29 mg/g, while the average content of amygdalin was 18.5, 17.7, 31.5, and 30.9 mg/g in Ph, Pj, Pp, and Pt, respectively. All the above information suggests that small seeds might be superior sources of Pruni Semen. This is the first comprehensive report on the identification of chemical components in Pruni Semen from different species.