Civilization IV: Colonization contains sixteen civilizations and twenty leaders:
| Civilization
| Leader
| Traits
|
| England
| King of England
| n/a
|
| France
| King of France
| n/a
|
| Netherlands
| King of Netherlands
| n/a
|
| Spain
| King of Spain
| n/a
|
| New England
| John Adams
| Libertarian, Tolerant
|
| George Washington
| Disciplined, Tolerant
|
| New France
| Samuel de Champlain
| Enterprising, Cooperative
|
| Louis de Frontenac
| Militaristic, Cooperative
|
| New Netherlands
| Adriaen van der Donck
| Charismatic, Mercantile
|
| Peter Stuyvesant
| Industrious, Mercantile
|
| New Spain
| Simon Bolivar
| Determined, Conquistador
|
| Jose de San Martin
| Resourceful, Conquistador
|
| Apache
| Mangas Coloradas
| Impressionable, Indulgent
|
| Arawak
| Agueybana
| Impressionable, Gracious
|
| Aztec
| Montezuma
| Prosperous, Gracious
|
| Cherokee
| Oconostota
| Impressionable, Gracious
|
| Inca
| Huayna Capac
| Prosperous, Mentor
|
| Iroquois
| Logan
| Indulgent, Mentor
|
| Sioux
| Sitting Bull
| Impressionable, Mentor
|
| Tupi
| Cunhambebe
| Prosperous, Indulgent
|
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