The Layton Art Gallery is a defunct art museum in Milwaukee, Wisconsin. Built at the initiative of British-American businessman Frederick Layton, the gallery was inaugurated in 1888 as the first public art institution in the city. Its one-story building, designed in the Greek Revival style by Scottish architect George Ashdown Audsley, stood at the corner of Mason and Jefferson streets, in downtown Milwaukee. The bulk of the gallery's works consisted of Layton's personal collection of European and American paintings and sculpture, assembled during the five years preceding the institution's opening, as well as subsequent purchases through an endowment.

Following Layton's death, art educator Charlotte Partridge opened the Layton School of Art in the basement of the gallery, a decision originally met with opposition from part of the public. Nevertheless, the school operated on site until 1951, when it relocated to a new building in the East Side district of Milwaukee. In 1957, the Layton Art Gallery merged with another institution, the Milwaukee Art Institute, to form the future Milwaukee Art Museum, housed in the County War Memorial designed by architect Eero Saarinen. The vacant Audsley building was razed in fall of that year. The original Layton Art Collection was entrusted to the new museum yet has remained under the purview of a distinct board of trustees since then.

Beginning in the 1870s, the idea of establishing a public art gallery was increasingly supported by Milwaukee's city leaders, along with the need for a permanent exhibition venue. Significant artworks in town were mostly confined to private residences, including the homes of collectors Martha Reed Mitchell and William H. Metcalf. Occasional attempts were made to provide a permanent venue for the display of art, including with the construction of the Milwaukee Industrial Exposition Building. Inaugurated in 1881,the structure was modeled after London's Crystal Palace and Philadelphia's Centennial Exposition's Main Building, and hosted annual exhibitions of art and industry. The building was destroyed by fire on June 4, 1905. Its ruins were razed and replaced by the Milwaukee Auditorium in 1909.

According to a story reprised by Frederick Layton himself, he and railroad magnate Alexander Mitchell took part in a dinner at the Milwaukee Club in 1883 to celebrate their imminent departure to Europe, upon which Layton commented that an art gallery was needed for the city of Milwaukee. Word spread quickly, with Layton called on the next day by a reporter about his plans to build the structure. Soon, the Milwaukee Sentinel reported that Layton "was now going abroad and intends studying the architecture and management of art institutes while there and hoped to pick up some information that would be of value in the construction of a model building." The information was reprinted in national newspapers such as The New York Times, persuading Layton to act on it.

While abroad, Layton hired George Ashdown Audsley, a Liverpool-based architect, to design plans for gallery building. Milwaukee architect Edward Townsend Mix worked jointly with his British counterpart to carry out the construction. The resulting design was a single-story top-lit gallery that differed from many other American gallery designs of the period, instead directly inspired by British galleries, including the Walker Art Gallery in Liverpool, the Dulwich Picture Gallery in London, and the Fitzwilliam Museum in Cambridge. The gallery's entrance was designed as a grand portico of simplified fluted Corinthian columns, with a frieze and facade ornaments made of terracotta, while the three remaining exterior walls were to be built using local Cream City brick. The project broke ground in October 1885.

Meanwhile, when collecting works of art, Layton sought out a range of popular artists of his time. He attended the New York estate sales of Alexander Turney Stewart and Mary J. Morgan, at which he purchased landscape scenes by painters John Constable and Régis François Gignoux, then crossed the Atlantic to pursue his acquisitions in Europe. A great number of Layton's purchases came from fine art dealer Arthur Tooth & Sons in London.

The Layton Art Gallery was officially inaugurated on April 5, 1888. The total cost for construction amounted to $115,000 (roughly $3.8 million in 2025 dollars, adjusted for inflation), to which Layton added a $100,000 endowment for the purchase of art and care of the building.

Among artists represented in Layton's inaugural gift were painters William-Adolphe Bouguereau, James Tissot, and Eastman Johnson (The Old Stagecoach, 1871). Over the next decades, purchases and gifts from local collectors including Frederick Pabst, Philip Danforth Armour, Edward Phelps Allis, Patrick Cudahy, William Plankinton, John Lendrum Mitchell, and the Vogel family brought in works by Winslow Homer, Jules Bastien-Lepage (Le Père Jacques, 1881), Frederic Leighton, Albert Bierstadt, Lawrence Alma-Tadema, Thomas Moran, Abbott Handerson Thayer, Mihály Munkácsy, and Sofonisba Anguissola. In 1893, Italian sculptor Gaetano Trentanove, a participant to the World's Columbian Exposition held in Chicago, completed a bust of Layton, while his entry into the world's fair, a marble sculpture titled The Last of the Spartans, was acquired for the gallery.