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Establishing accurate genetic similarity and dissimilarity between individuals is an essential and decisive point for clustering and analyzing inter and intra population diversity because different similarity and dissimilarity indices may yield contradictory outcomes. We assessed the variations caused by three commonly used similarity coefficients including Jaccard, Sorensen-Dice and Simple matching in the clustering and ordination of seven Iranian native silkworm, Bombyx mori L. (Lepidoptera: Bombycidae), strains analyzed by amplified fragment length polymorphism markers. Comparisons among the similarity coefficients were made using the Spearman correlation analysis, dendrogram evaluation (visual inspection and consensus fork index - CIC), projection efficiency in a two-dimensional space, and groups formed by the Tocher optimization procedure. The results demonstrated that for almost all methodologies, the Jaccard and Sorensen-Dice coefficients revealed extremely close results, because both of them exclude negative co-occurrences. Due to the fact that there is no guarantee that the DNA regions with negative cooccurrences between two strains are indeed identical, the use of coefficients such as Jaccard and Sorensen-Dice that do not include negative co-occurrences was imperative for closely related organisms.