Nuts and seeds are well known for their health benefits due to their bioactive compounds, including phytosterols. However, information on phytosterols in nuts and seeds is scare. In this study, the free and esterified phytosterols in 65 nut and seed samples from China were analyzed using solid-phase extraction combined with gas chromatography–mass spectrometry. The compositions of phytosterols were compared and classified by hierarchical cluster analysis. Results showed that the total phytosterols ranged from 15.9 mg/100 g dry weight of chestnuts to 255.2 mg/100 g dry weight of pistachios, and approximately 54.1%–77.2% of the total phytosterols were in the free form. Moreover, 76.1%–96.4% of the total phytosterols belonged to 4-desmethyl sterols, and more than 92.6% belonged to plant sterols. In most nuts and seeds, β-sitosterol was the major phytosterol, accounting for 62.4%–87.1%, except that in pumpkin seed kernel and watermelon seed kernels, the major phytosterol was α-spinasterol (57.7% and 67.8%, respectively). The nut and seed samples were classified into four groups on the basis of the composition of phytosterols. This study revealed the difference of phytosterols between nuts and seeds, which may contribute to their specific health benefits.