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Calotropis procera is a soft-wooded, evergreen, perennial shrub species which grows in a variety of environments including dry habitats such as the Brazilian semi-arid region. This study aimed to evaluate the variation in the oil content and fatty acid composition of C. procera seeds from four climatically different locations in the state of Pernambuco in the northeast of Brazil. The seed oil content of C. procera ranged from 19.7 to 24.0 %. Five main fatty acids were identified, with a predominance of unsaturated linoleic and oleic acids (approximately 70 %). Our results suggesting that environmental conditions influenced the seed oil biosynthesis of C. procera, because the localities with high temperature and low precipitation had increased oil content and total biosynthesis of saturated fatty acids. Multivariate analysis showed high similarity among three localities which share certain physiographic characteristics and climatic conditions such as temperature and precipitation values. The oil profile of C. procera presents interesting features that highlight its potential as a future alternative for the biodiesel market, especially in semi-arid regions.