Enterocytozoon bieneusi was detected in dogs in Bogota, Colombia for the first time. Of 120 dogs, 15% were positive by polymerase chain reaction (PCR). Infected dogs ranged from 2 to 14 years of age, and more male than female dogs were infected with E. bieneusi (20% versus 5%). All PCR-positive specimens were sequenced and three genotypes were identified. A dog-specific genotype (PtEbIX) was identified in feces from 16 dogs. For two dogs, the nucleotide sequences obtained were genetically identical to the E. bieneusi Peru 5 and K genotypes previously reported as human pathogens. This is the first time that Peru 5 and K genotypes have been identified in dogs. This study confirms that dogs are infected with both human pathogenic and host-specific genotypes.