The spectacled bear (Ursidae: Tremarctos ornatus) is an emblematic umbrella species and one of the top carnivores in the Andean mountains. It is also listed as vulnerable by IUCN and as endangered by CITES. We analyzed the genetic structure of this species in nine geographical regions representing the three Andean Cordilleras in Colombia. We sequenced six mitochondrial genes in 115 spectacled bears; a subset of these specimens (n = 61) were genotyped at seven nuclear microsatellites. We addressed three objectives: 1) determine the genetic diversity and historical demographic changes of the spectacled bear in Colombia; 2) determine phylogeographic patterns of genetic divergence among spectacled bear populations in Colombia; and 3) estimate the levels of gene flow among different regions of Colombia. Our analyses show evidence of high mitochondrial genetic diversity in spectacled bears, both in Colombia as well as in each of the nine regions, most particularly Norte de Santander, Nariño, and Antioquia-Córdoba. In addition, we detected population expansion in Colombia that occurred around 24,000 years ago, followed by a population decrease during the last 7,000 years, and a sudden expansion in the last 300 years. Phylogenetic analyses showed few well-supported clades, with some haplotypes detected in all the departments and Colombian Andean Cordilleras, and other haplotypes restricted to certain geographical areas (Antioquia, Norte de Santander, Cundinamarca, and Nariño). We detected significant genetic heterogeneity among some departments and among the three Colombian Andean Cordilleras for both mitochondrial and nuclear genes. Nevertheless, the moderate levels of gene flow estimated from F_ST statistics suggest that geographical barriers have not been definitive obstacles to the dispersion of the spectacled bear throughout Colombia. Despite these gene flow estimates, significant spatial autocorrelation was detected for spectacled bear in Colombia, where two kinds of spatial patterns were discovered: genetic patches of 144 km of diameter, and isolation by distance among bears separated from 578 to 800 km. The two most northern spectacled bear populations of Colombia (Norte de Santander and Antioquia) also were the two most differentiated. Their distinctiveness may qualify them as distinct Management Units (MUs) in the context of conservation policies for the spectacled bear in Colombia.