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Genetic differentiation between 40 lentil genotypes was tested using molecular markers. The genotypes were produced from a Greek landrace of commercial interest via the honeycomb breeding methodology, i.e., single-plant selection in the absence of competition, across three successive pedigree generations. The selected genotypes from each generation were examined for genetic relationships using 15 SSR molecular markers with HRM analysis. As expected, low variation among consecutive generations at the level of 2.5–7.7% was detected. Analysis of molecular variance (AMOVA) revealed that partitioning of this variation was at higher percentage within each generation’s population than between them. Population structure analysis indicated that ongoing selection could effectively shift the allelic composition in each generation. The applied honeycomb breeding methodology that effectively improved progeny yield and seed quality increased the percentage of favorable alleles altering allelic composition but not eliminating genetic variation of the breeding population.

Genetic Variation of a Lentil (Lens culinaris) Landrace during Three Generations of Breeding