Pathogen variation plays an important role in the dynamics of infectious diseases. In this study, the genetic variation of 279 Phytophthora infestans isolates was assessed using a combination of 12 microsatellite simple-sequence repeat markers. Isolates were collected from 11 different potato cultivars in 11 different geographic localities of the central region of Colombia. The objective of this study was to determine whether populations were differentiated by host genotype or geographic origin. Within a single clonal lineage, EC-1, 76 genotypes were detected. An analysis of molecular variance attributed most of the variation to differences within host genotypes rather than among the host genotypes, suggesting that host cultivars do not structure the populations of the pathogen. Furthermore, the lack of a genetic population structure according to the host cultivar was confirmed by all of the analyses, including the Bayesian clustering analysis and the minimum spanning network that used the Bruvo genetic distance, which suggested that there are no significant barriers to gene flow for P. infestans among potato cultivars. According to the geographic origin, the populations of P. infestans were also not structured, and most of the variation among the isolates was attributed to differences within localities. Only some but not all localities in the north and west of the central region of Colombia showed some genetic differentiation from the other regions. The absence of sexual reproduction of this pathogen in Colombia was also demonstrated. Important insights are discussed regarding the genetic population dynamics of the P. infestans populations of the central region of Colombia that were provided by the results. In Colombia, there is a high genetic variation within the EC-1 clonal lineage with closely related genotypes, none dominant, that coexist in a wide geographic area and on several potato cultivars.