The Spanish installed Atahualpa's brother Manco Inca Yupanqui in power; for some time Manco cooperated with the Spanish, while the Spanish fought to put down resistance in the north. Meanwhile an associate of Pizarro's, Diego de Almagro, attempted to claim Cusco for himself. Manco tried to use this intra-Spanish feud to his advantage, recapturing Cusco in 1536, but the Spanish retook the city afterwards. Manco Inca then retreated to the mountains of Vilcabamba, Peru, where he and his successors ruled for another 36 years, sometimes raiding the Spanish or inciting revolts against them. In 1572 the last Inca stronghold was conquered, and the last ruler, Túpac Amaru, Manco's son, was captured and executed. This ended resistance to the Spanish conquest under the political authority of the Inca state.

After the fall of the Inca Empire, the new Spanish rulers brutally oppressed the people and suppressed their traditions. Many aspects of Inca culture were systematically destroyed, including their sophisticated farming system. The Spaniards used the Inca mita (mandatory public service) system to literally work the people to death. One member of each family was forced to work in the gold and silver mines, the foremost of which was the titanic silver mine at Potosí. When a family member died, which would usually happen within a year or two, the family would be required to send a replacement.

The effects of smallpox on the Inca empire were even more devastating. Beginning in Colombia, smallpox spread rapidly before the Spanish invaders first arrived in the empire. The spread was probably aided by the efficient Inca road system. Within a few years smallpox claimed between 60% and 94% of the Inca population, with other waves of European disease weakening them further. Smallpox was only the first epidemic. Typhus (probably) in 1546, influenza and smallpox together in 1558, smallpox again in 1589, diphtheria in 1614, measles in 1618 - all ravaged the remains of Inca culture.