Background The phylogenetic relationships and phylogeny of twenty-six Trichoderma species collected from various Egyptian locations were investigated. The genetic diversity among the examined isolates was tested using the rep-PCR marker. Trichoderma species were screened for their cellulase activities. Results Three isolates demonstrated highly significant FPase activities, namely MNF-MAS-Tricho 1, MNF-MAS-Tricho 2, and MNF-MAS-Tricho 3 (0.50, 0.39, and 0.49 IU ml −1 , respectively). MNF-MAS-Tricho1 showed the highest significant CMCase activity (0.80 IU ml −1 ). Concerning β-glucosidase, MNF-MAS-Tricho 1 was the highest (0.78 IU ml −1 ), while MNF-MSH-Trich 11 and MNF-MAS-Tricho 15 were the lowest (0.36 IU mL −1 ). The percentage of polymorphism ranged from 46.15 to 83.33%. (GTG)5 marker produced the greatest number of polymorphic loci (13 loci out of 18 loci) with about 83.33% polymorphism, followed by rep-10 with 69.2% polymorphism. Furthermore, the polymorphism information content (PIC) estimates ranged between 0.285 for Rep-10 and 0.340 for (GTG) 5 with an average of 0.306. The tested primers exhibited high discriminating and resolving powers. Conclusion The findings of this investigation were used to classify Trichoderma species, evaluate their genetic variability using ITS sequencing, rep-PCR, and measure their cellulase activities. These markers can facilitate more rapid and less complicated studies of Trichoderma population dynamics and evaluate their establishment after release into agricultural environments. The results will help to evaluate the genetic diversity of Trichoderma in future research.