Since 1924, the Gallery has been arranged on the second floor of the Pitti Palace and extends as far as the rooms an the facade used by the Medici for the palace library and the side wings used for children and retainers. It was founded in 1914 and initially comprised works of art brought in from the Academy of Fine Arts. The current museum collection comprise thirty rooms that trace a wide chronological arc: from the time of Pietro Leopoldo up to the First World War. The tour organised in chronological order and by historical-topical category, attempts to furnish the visitor with a clear view of the histories of the various core collections and enable a correct reading of the diverse atmospheres, marked as they are by the personal tastes of the royal families alternating in their reigns. Today it has a very special juridical nature due to a convention signed by the Italian State and the Municipal Administration of Florence.

The thirty rooms of the Gallery have recently been reorganised, according to chronological criteria, Down a period of time going from Neoclassicism (the age of Peter Leopold) to the 1920' s. The rooms on the second floor have been restored, but the decoration, upholstering and furniture of the Lorraine period have been maintained.

The itineray begins with both Neoclassic works like the "Oath of the Saxons to Napoleon" by Pietro Benvenuti and romantic works like the grandiose "Entry of Charles VIII" by Giuseppe Bezzuoli or "The two Foscari" by Francesco Hayez. There are also many fine sculptures of the same period like "Calliope" of Antonio Canova, "Psyché" by Pietro Tenerani and the famous "Abel" by Giovanni Dupré. The collection includes a vast assortment of paintings based on historical subjects that document one of most significant aspects of the first half of the 19th century culture. These comprise works by Sabatelli, Pollastrini or by Stefano Ussi with his famous "Expulsion of the Duke of Athens". Yet the paintings that most characterise the Gallery are those of the Macchiaioli, the famous Tuscan artists of the mid-19th century that set out the premise for a wide-scale innovation at a national level. This section comprises important works by Giovanni Fattori, like the "Rotonda Palmieri", the "Battle of Magenta", the “Staffato”, and a rich series of landscapes and scenes of life in the Maremma (the "Market in Maremma", the "Ox cart", the "Salto delle pecore"). Many of the works of these artists displayed in the Gallery belong to the collection of Diego Martelli, a critic and friend of the Macchiaioli who left their paintings to the museum at the end of the last century.

There are also many paintings by Silvestro Lega and Telemaco Signorini with views and interior scenes, while Giovanni Boldini is represented with a series of his rapid and elegant portraits. The sculptures of this section include the works by Adriano Cecioni, who lucidly translated and experimented the tonal ideas prevalent to whom the touch was so important.

In addition to the above-mentioned collections belonging to the early and late 19th century, the Museum also displays a lavish collection of 19th century works that will be arranged in the so-called "Mezzanino degli Occhi" (Mezzanine of the Eyes, the “eyes” being windows in the shape of a circle.