Spinach (Spinacea oleracea L.) is a valuable agricultural crop that accumulates phytoecdysteroids, polyhydroxylated triterpenoids, which may play a role in plant defense and have purported health benefits for human consumers. In this study, phytoecdysteroid accumulation was measured in seeds and shoots of 15 spinach accessions to determine whether phytoecdysteroid levels vary between spinach varieties and whether seed content could reliably predict relative levels in the edible foliage. Additionally, phytosterols, precursors to phytoecdysteroids, were examined to determine potential points of regulation of spinach phytoecdysteroid biosynthesis. Significant variations in phytoecdysteroid levels between accessions were observed (P < 0.05), suggesting the potential for genetic manipulation through traditional breeding or genetic engineering to increase phytoecdysteroid levels in spinach. However, results suggest that estimation of phytoecdysteroid levels in shoots may not be achieved by measuring levels in the seeds. Levels of phytoecdysteroids in spinach ranged from 19.9 to 44.1 μg per shoot, 0.7 to 1.2 μg·mg−1 dry mass shoot, 3.2 to 9.6 μg per seed, and 0.5 to 1.1 μg·mg−1 seed. Several phytosterols connected to the phytoecdysteroid biosynthetic pathway were identified by gas chromatography–mass spectroscopy, predominantly spinasterol, 5-dihydroergosterol, and 22-dihydrospinasterol, which comprised 79.8%, 6.3%, and 4.6% of the total phytosterol content, respectively. Detection of the phytosterols cycloartenol and lanosterol in spinach suggests that spinach may also have dual biosynthetic pathways to phytosterols that contribute to the production of phytoecdysteroids.