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The Cathedral of Florence is one of the largest churches in the world and is known for one of the most famous domes ever, which characterizes the skyline of the city. The dimensions of the building mean that the dimensions of the roof are equally large and so are the wooden structures that support it. The roof of the cathedral is organized on two levels: the roof of the large central nave and, at a lower level, those of the two lateral naves. The purpose of this paper is the identification of the wood species of which the structures are made. The sampling method of the 408 samples that have been identified is then described, the methods followed to reach a reliable identification and finally the results. The timbers most represented among the structural elements are those of silver fir, chestnut and elm. Other timbers are then found in the other components less directly linked to the main structural parts that make up the trusses. The paper then discusses the technological implications on the use of those woods within the wooden covering structures of the cathedral and the main sources of timber that the builders had available, in particular the Casentino forests that the Municipality of Florence had donated to the structure that managed the construction of the cathedral (Opera di Santa Maria del Fiore—OPA). OPA still exists today and is responsible for the maintenance of the cathedral and other annexed buildings. [ABSTRACT FROM AUTHOR]