This article analyzes the demographic development of the Province of Tunja during the first century of Spanish domination. An effort is made towards estimating the size of the indigenous population at the time of Conquest, while at the same time evaluating the decrease in population for the following hundred years. The author carefully examines the studies on historical demography for the Colombian Eastern Highlands, adding new sources and methodological considerations to the debate. It is argued that while native populations from other parts of Latin America were able to recover from this initial crisis during the first decades of the 17th century, this never happened in the province of Tunja and the population continued to diminish during this century due to the impact of contagious diseases, harsh treatment, excessive work and migration.