The El Greco Museum is a house-museum dedicated to the Mannerist painter Domenikos Theotokopoulos, who was born in Crete but produced most of his work in Toledo. The institution, which was opened in 1911 to collect works by the Cretan artist, is a recreation of the one the painter used to work in. The museum, which is part of the Ministry of Culture and Sport, is managed exclusively by the Directorate General of Fine Arts.

The museum conserves approximately twenty canvases by El Greco, including the Apostles, which is considered to be of the highest quality of all those preserved, one of the best versions of The Tears of Saint Peter, the portraits of Diego de Covarrubias and his brother Antonio, the Altarpiece of Saint Bernardino, on loan from the Prado Museum, and the View and Plan of Toledo, perhaps the most significant piece in the institution. Likewise, in 2021, the Ministry of Culture acquired the Crucifixion for the El Greco Museum, the first painting by the Cretan to be incorporated into the art gallery since its foundation in 1910.

The museum also has a notable repertoire of works by Luis Tristán, considered El Greco's best disciple, in whose work he combined the strong imprint of his master with that of the Caravaggist naturalism that he came to know during his training period in Rome. Other artists represented include Jorge Manuel Theotocópuli, the son of El Greco - and of whom a copy of Expolio, owned by the Prado, is on display - as well as Sánchez Coello, Bartolomeo Passerotti, Herrera the Elder, Juan Bautista del Mazo (with a superb portrait of Queen Mariana of Austria dressed in widow's headdresses, which was a gift from Huntington to the Marquis de la Vega-Inclán), Valdés Leal, Joaquín Sorolla (Portrait of the Marquis de la Vega-Inclán) and Raimundo de Madrazo. Many of these works were destined for the "Museum of Spanish Art", a project conceived by the Marquis which never materialised and which would serve to contextualise El Greco's work.

At least 25 of these works have been loaned by the Museo del Prado.