The Gallery has a large and varied collection of paintings, drawings, sculpture and photographs. Although its focus is on Canadian art, it holds works by many noted American and European artists. It has a strong contemporary art collection with some of Andy Warhol's most famous works. In 1990 the Gallery bought Barnett Newman's Voice of Fire for $1.8 million, igniting a storm of controversy. Since that time its value has appreciated sharply. In 2005, the Gallery acquired a painting by Italian Renaissance painter Francesco Salviati for $4.5 million. Its most famous painting is likely The Death of General Wolfe by Anglo-American artist Benjamin West.
In 2005, a sculpture of a giant spider, Louise Bourgeois's Maman, was installed in the plaza in front of the Gallery. In 2011 the gallery installed Canadian sculptor Joe Fafard's Running Horses next to the Sussex Drive entrance, and American artist Roxy Paine's stainless steel sculpture One Hundred Foot Line in Nepean Point behind the gallery.
The Gallery organizes its own exhibits which travel across Canada and beyond, and hosts shows from around the world, often co-sponsored with other national art galleries and museums.
The Gallery's collection has been built up through purchase and donations. Much of the collection was donated, notably the British paintings donated by former Governor General Vincent Massey and that of the Southam family.
The museum features Canadian, Native and Inuit art, American and European painting, sculpture, prints and drawings, modern and contemporary art and photographs. The largest work in the Gallery is the entire interior of the Rideau Street Chapel, which formed part of the Convent of Our Lady Sacred Heart, The interior decorations of the Rideau Street Chapel were designed by Georges Couillon in 1887. After the convent was demolished in 1972, the chapel was dismantled, stored and reconstructed within the gallery as a work of art in 1988.