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Single nucleotide polymorphism (SNP) markers have multiple applications in plant breeding of small grains. They are used for the selection of divergent parents, the identification of genetic variants and marker-assisted selection. However, the use of SNPs in varietal purity assessment is under-reported, especially for multi-line varieties from the public sector. In the case of variety evaluation, these genetic markers are tools for maintaining varietal distinctness, uniformity and stability needed for cultivar release of multi-line or pure-line varieties of inbred crops. The objective of this research was to evaluate the purity and relationships of one original (AV-25) and two multi-line sub-populations (AV25-T and AV25-S) of the inbreeding species, oats (Avena sativa L.). Both sub-populations could be useful as forages in the central highland region of Colombia (>2000 masl), such as in the departments of Boyacá and Cundinamarca, even though they were derived from an original composite mixture widely used in the mountainsides of the southern department of Nariño named Avena 25. Representative single plant selections (SPS) from the two sub-populations were grown together with SPS harvests from off-type plants (early and late) and plants from the original AV25 composite mixture, to determine their genetic similarity. Plants were genotyped by DNA extraction of a plateful of 96 individual plant samples and SNPs were detected for an Illumina Infinium 6K Chip assay. The data were used for the analysis of genetic structure and population relationships. The grouping observed based on the genetic data indicated that AV25-T and AV25-S were homogeneous populations and somewhat divergent in their genetic profile compared to the original AV25-C mix. In addition, to the two commercial, certified oat varieties (Cajicá and Cayuse) were different from these. The early and late selections were probable contaminants and could be discarded. We concluded that the use of SNP markers is an appropriate tool for ensuring genetic purity of oat varieties.