Royal Botanical Gardens (RBG) is headquartered in Burlington and also include lands in Hamilton, Ontario, Canada. It is one of the major tourist attractions between Niagara Falls and Toronto, as well as a significant local and regional horticultural, education, conservation and scientific resource. On 31 July 2006, Royal Botanical Gardens was selected as the National Focal Point for the Global Strategy for Plant Conservation (GSPC) by Environment Canada.

The 980 hectares (2,422 acres) of nature sanctuary owned by Royal Botanical Gardens is considered the plant biodiversity hotspot for Canada, with a very high proportion of the wild plants of Canada in one area; is an Important Bird Area according to Bird Studies Canada; and is part of the the Niagara Escarpment World Biosphere Reserve. More than 1,100 species of plants grow within its boundaries including the Bashful Bulrush (Trichophorum planifolium) which is found nowhere else in Canada, and the largest remaining population of Canada's most endangered tree, the Red Mulberry (Morus rubra). Both of these plants are listed as Endangered in Canada under the Species at Risk Act.

Initial sections of the RBG were built during the Great Depression in the 1930s as a make work project, under the impetus of Thomas McQuesten. It beautified derelict or undeveloped land in east Hamilton and west Burlington. For instance, a disused gravel pit was turned into the Rock Gardens, by using stone relocated from the Niagara Escarpment. The original vision of the RBG was a mixture of horticultural displays, and protected natural forests and wetlands. Royal Assent was obtained in 1930 to call the gardens "Royal Botanical Gardens".

The first Director of RBG, Dr. Norman Radforth, was appointed in 1947 and was a Professor of Botany at nearby McMaster University. In the early 1950s, Dr. Leslie Laking was appointed as Director, and served until the early 1980s. Under his guidance the institution developed into the major entity it is today. With approximately 1,100 ha (2,700 acres) of property, Royal Botanical Gardens is one of the largest such institutions in North America. In 2006 the Auxiliary of RBG published Love, sweat and soil: a history of Royal Botanical Gardens from 1930 to 1981 authored by Dr. Laking.

Other attractions at RBG include the Arboretum, the outdoor education centre, a network of trails and outdoor floral arrangements. Some of the trails link to the Bruce Trail, and a building in the RBG Arboretum, Raspberry House, is the headquarters of The Bruce Trail Conservancy.

The natural lands or nature sanctuaries of Royal Botanical Gardens include some of the most significant wildlife and native plant areas in Canada. The largest area, which includes the wetland called Cootes Paradise or Dundas Marsh, is a major spawning area for native fish species in Lake Ontario, one of the best bird-watching areas in Canada, and is the subject of one of Canada's largest ecological rehabilitation efforts aimed at a wetland, 'Project Paradise.'

The Royal Botanical Gardens holds the second-largest garden show in Canada simply called The Ontario Garden Show. It comes second only to Canada Blooms which is held in Toronto.[citation needed]

Innovative educational programs are operated from both RBG's main building in Burlington and the Nature Interpretive Center, located in the Arboretum to the north of Cootes Paradise. Over 18,000 school children per year visit the organized school programs, and over 200 public education offerings include such diverse topics as botanical illustration, organic cooking and basic botany. Aldershot School offers an ECO Studies program in conjunction with the RBG.

Royal Botanical Gardens is a member of the American Public Gardens Association, and is very active in local, regional, national and international efforts to conserve plant diversity and ensure its sustainable use. In 2006 RBG was named by Environment Canada as the National Focal Point for Canada for the Global Strategy for Plant Conservation (GSPC), an element of the UN's Convention on Biological Diversity.