When the conquistadors arrived in Yucatán from the 1530s onwards, Maya towns and provinces were still vigorously independent, as the Spanish found to their cost on several occasions. Northern Belize was part of the wealthy Maya province of Chactemal (the name lives on today as Chetumal, in Quintana Roo, Mexico), with its capital probably at Santa Rita , near Corozal. Trade, alliances and wars kept Chetumal in contact with surrounding Maya states up to and beyond the Spanish conquest of Aztec Mexico. Further south was the province known to the Maya of Chetumal as Dzuluinicob - "land of foreigners" - whose capital was Tipu , located at Negroman, on the Macal River south of San Ignacio. The Maya here controlled the upper Belize River valley and put up strenuous resistance to attempts by the Spanish to subdue and convert them. The struggle was to continue with simmering resentment until 1707, when the population of Tipu was forcibly removed to Lago de Petén Itzá, near Tikal.

The first Europeans to set eyes on the mainland of Belize were Spanish sailors in the early 1500s, but they didn't attempt a landing. In 1511 a small group of shipwrecked Spanish sailors managed to reach land on the southern coast of Yucatán: five were immediately sacrificed but the others were taken as slaves. One of them, Gonzalo Guerrero , later married the daughter of Nachankan, the chief of Chetumal, and became a crucial military adviser to the Maya in their subsequent resistance to Spanish domination. The archeologist Eric Thompson calls him the first European to make Belize his home.

By 1544 however, Gaspar Pacheco had subdued Maya resistance sufficiently to found a town on Lake Bacalar, and a mission was established at Lamanai in 1570. Maya resentment was always present beneath the surface, however, and boiled over into open rebellion in 1638, forcing Spain to abandon the area. The whole region from southern Yucatán to Honduras was never completely pacified by the Spanish, nor were administrative boundaries clearly defined, but it is likely that the Maya of Belize were influenced by the Spanish, even if they were not ruled by them.