The first nucleus of the Vatican Museums was the collection of full-sized statues from Julius II displayed in the so-called "Courtyard of Statues", which today is known as the Octagonal Courtyard. These art collections are currently housed together and made available to the public in different buildings within the museum.

The Papal Museums and Galleries originated from the work of Clement XIV and Pius VI, and for this reason the name originally was the Pio Clementine Museum. Pius VII notably expanded it, adding the Chiaramonti Museum, the Braccio Nuovo and the Lapidary Gallery.

Gregory XVI founded:

the Etruscan Museum (1837) with relics recovered from excavations in southern Etruria beginning from 1828;

the Egyptian Museum (1839) with original Egyptian works from explorations in Egypt, as well as other pieces found in the classical art collection, in the Capitoline Museum and the Lateran Profane Museum (1844). These included bas-reliefs, statues, and mosaics of the Roman period that were not able to be placed in the Vatican Palaces.

In 1854 Pius IX added the Pio Christian Museum to the Lateran Profane Museum, which is made up of sculptures, especially sarcophagi, and ancient Christian inscriptions, and later adding two rooms of artifacts taken from the excavations of Ostia carried out in San Ercolano and San Aurea (1856-1869), as well as from Prince A. Torlonia in Porto in 1866. Under the pontificate of Saint Pius X, in 1910, a section was added containing 137 inscriptions from ancient Hebrew cemeteries in Rome, in a large part from the cemetery on Via Portuense, donated by the proprietors of the foundation of Marquees Pellegrini-Quarantotti. These two collections were transported, at the request of John XXIII, from the Lateran Palace to a new specially erected building in the Vatican; in 1970 these collections were reopened to the public, with the name Gregorian Profane Museum and Pio Christian Museum..

The Museum is made up of several other sections:

the Gallery of Tapestries, a collection of tapestries of different fabrics from the 16th and 17th centuries;

the Gallery of Maps, instituted by Gregory XIII and restored by Urban VIII;

the Sobieski Room as well as the Room of the Immaculate Conception;

the Stanze and the Loggia of Raphael, commissioned by Julius II and Leon X;

the Chapel of Nicholas V, painted by Fra Angelico;

the Sistine Chapel, named for its founder, Sixtus IV;

the Borgia Apartment, home of Alexander VI, restored and opened to the public by Leon XIII in 1897;

the Vatican Picture Gallery, placed in the Borgia Apartment under Pius VII , then in the Third Loggia by Gregory XIII. In 1909 it was taken by Saint Pius X to the Gallery underneath the wing of the Library that faces the gardens, and finally put in a special building by Pius XI in 1932;

the Missionary Ethnological Museum was founded by Pius XI in 1926, and arranged in the upper floors of the Lateran Palace; it was later transported, at the request of John XXIII, into the Vatican, where it was reopened to the public in the same building that housed other collections formerly of the Lateran Palace. In 1973 the Collection of Modern Religious Art was added, and inaugurated by Paul VI on June 23 of that year.

The Historical Museum, founded also in 1973, was transferred to the Papal Apartment of the Lateran Palace in 1987, where a collection of portraits of the Popes was included; the relics of the Pontifical Military Corps as well as artifacts from the Pontifical Chapel and Family were added. And finally, a documentation of ceremonies which are no longer in use, are stored as well in this section. The Carriage Museum, a section of the Historical Museum was also set up by Paul VI in 1973 and houses the Popes' carriages and automobiles.

Before the Lateran Accords, the Pontifical Museums and Galleries were part of the SS. PP AA. Prefecture; since 1929 they have been placed under the Governatorate of the Vatican City State.