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By the 16th century, the earliest time for which there is a historical record, major Native American groups included the Apalachee of the Florida Panhandle, the Timucua of northern and central Florida, the Ais of the central Atlantic coast, the Tocobaga of the Tampa Bay area, the Calusa of southwest Florida and the Tequesta of the southeastern coast.

Florida was the first region of the continental United States to be visited and settled by Europeans. The earliest known European explorers came with the Spanish conquistador Juan Ponce de León. Ponce de León spotted and landed on the peninsula on April 2, 1513. He named it La Florida in recognition of the verdant landscape and because it was the Easter season, which the Spaniards called Pascua Florida (Festival of Flowers). The following day they came ashore to seek information and take possession of this new land. The story that he was searching for the Fountain of Youth is mythical and appeared only long after his death.

In May 1539, Conquistador Hernando de Soto skirted the coast of Florida, searching for a deep harbor to land. He described a thick wall of red mangroves spread mile after mile, some reaching as high as 70 feet (21 m), with intertwined and elevated roots making landing difficult. The Spanish introduced Christianity, cattle, horses, sheep, the Castilian language, and more to Florida. Spain established several settlements in Florida, with varying degrees of success. In 1559, Don Tristán de Luna y Arellano established a settlement at present-day Pensacola, making it the first attempted settlement in Florida, but it was mostly abandoned by 1561.

In 1565, the settlement of St. Augustine (San Agustín) was established under the leadership of admiral and governor Pedro Menéndez de Avilés, creating what would become one of the oldest, continuously-occupied European settlements in the continental U.S. and establishing the first generation of Floridanos and the Government of Florida. Spain maintained strategic control over the region by converting the local tribes to Christianity. The marriage between Luisa de Abrego, a free black domestic servant from Seville, and Miguel Rodríguez, a white Segovian, occurred in 1565 in St. Augustine. It is the first recorded Christian marriage in the continental United States.