The aim was to establish the genetic diversity and population structure of three guinea pig lines, from seven production zones located in Nariño, southwest Colombia. A total of 384 individuals were genotyped with six microsatellite markers. The measurement of intrapopulation diversity revealed allelic richness ranging from 3.0 to 6.56, and observed heterozygosity (Ho) from 0.33 to 0.60, with a deficit in heterozygous individuals. Although statistically significant (p < 0.05), genetic differentiation between population pairs was found to be low. Genetic distance, as well as clustering of guinea-pig lines and populations, coincided with the historical and geographical distribution of the populations. Likewise, high genetic identity between improved and native lines was established. An analysis of group probabilistic assignment revealed that each line should not be considered as a genetically homogeneous group. The findings corroborate the absorption of native genetic material into the improved line introduced into Colombia from Peru. It is necessary to establish conservation programs for native-line individuals in Nariño, and control genealogical and production records in order to reduce the inbreeding values in the populations.