In order to determine the accuracy of species identification of the eight Trichoderma strains, DNA barcode study through intraspecific and interspecific gap analysis was conducted. Comparing the ITS1 region to the Tef1 region, it was found that ITS1 region provided a clear, distinguishable barcode, showing that the ITS1 primer is more effective at differentiating between different species in our comparable set of sequences than Tef1. Also, the eight strains were subjected for genetic diversity study with 12 ISSR primers where high levels of genetic diversity was indicated by high polymorphism and Nei's genetic diversity, with h of 0.46. Plant growth promoting attributes were further tested for eight Trichoderma strains which confirmed three of the strains—Trichoderma harzianum MC2, Trichoderma harzianum NBG, and Trichoderma lentiforme JC1—to exhibit the highest levels of PGPR characteristics. The research demonstrates the genetic diversity of the eight Trichoderma strains and viability of using the strains for production of biofertilizer in sustainable agriculture.