Teak (Tectona grandis L.f.), a paragon timber tree of tropical deciduous forests of Central and Peninsular India, is highly prized for its wood colour, decorative grains, durability and lightness. An experiment was carried out to compare the genetic variation detected and genetic relationships inferred in five teak populations via 10 genomic DNA samples per population each of either single seed or bulk of 3- or 5- seeds with the help of ISSR markers. The genomic DNA of single seed exhibited higher number of polymorphic loci, per cent polymorphism, nei’s genetic diversity and shannon Information Index than the bulk genomic DNA of 3- or 5- seeds. The bulking of genomic DNA of 3- and 5- seeds using Nei’s genetic distance coefficient revealed similar genetic relationships, which were at variance with those in single seed treatment. Mantel’s correlation test among the genetic distance matrices of single seed sampling, 3-seed bulk and 5-seed bulk sampling also confirmed the trend. Since the bulking of genomic DNA did not generate compatible estimates of diversity parameters and genetic relationship of five populations from its single seed sampling, we recommend strict guarding of identities of genotypes within the collected samples for obtaining precise estimates and drawing accurate conclusions about the genetic diversity and clustering of populations.