The purpose of the study was to gain insight into the genetic variability of the Letelle sheep breed, a breed that has been managed as a closed population for 90 years, with no new genetic material being permitted into the breed. The Letelle is a South African developed dual-purpose sheep breed and is classified as a Merino type with a Spanish Merino origin. The breed exhibits good fine wool characteristics and yields high-quality mutton. Line-breeding, family-breeding, and inbreeding are applied, and multiple sire matings are practised to prevent a sire from having a large influence on the national flock. Ear samples were collected from 10 animals each from 10 commercial and 11 seed-stock flocks and genotyped using 17 microsatellite markers. Unbiased heterozygosity ranged from 0.58 to 0.68 and the observed heterozygosity from 0.52 to 0.65. The estimated effective population size (Ne) was 228.2 - 321.9. Results from analysis of molecular variance (AMOVA), a Bayesian assignment test, and a neighbour-joining (NJ) tree suggested that no genetic sub-structure existed within this population and that the seed-stock and commercial flocks could be regarded as one genetic population. The average within flock (FIS) and within breed (FIT) inbreeding coefficients were 10.1% and 14.5%, respectively. Despite the level of inbreeding, levels of genetic diversity were moderate and potentially provide opportunities for future selection and adaptation. Further testing could identify flocks in which conservation management is required as well as those with high genetic variability, which would provide the best reservoir for selection to adapt to future climatic challenges.