Abstract Background The phylogeny and phylogenetic relationships of Trichoderma species were investigated by many methods including maximum parsimony and distance analysis of DNA sequences from multiple genetic loci. 5.8S rDNA sequence analysis is a powerful tool in evolution studies. Main body The aim of this study was to identify and determine the genetic distance between 36 Trichoderma isolates from Egyptian and Saudi Arabian soils based on their 5.8S rDNA sequences. The genetic diversity of Trichoderma (Hypocreales, Ascomycota) species from Egypt and Saudi Arabia was investigated. The DNA barcoding of 36 isolates, based on the internal transcribed spacers 1 and 4 (ITS1 and 4) of the ribosomal RNA gene cluster and the partial sequence of this gene, revealed the diversity of these isolates. BLAST query showed that two species, namely T. longibarchiatum (42.7% of the isolates) and T. lentiforme (33.3% of the isolates), pre-dominated the isolates. Variation among specimens of the same species was detected. Cluster analysis of the Egyptian isolates showed two groups; the first group was subdivided into two subgroups, the first of which included T. lentiforme and T. crassum isolates, and the second subgroup harbored T. longibrachiatum, T. pseudokoningii, and T. bissettii. The second group included T. neokoningii and T. yunnanens. The cluster analysis of isolates from Saudi Arabia also consisted of two groups. The first group was subdivided into two subgroups; the first included T. longibrachiatum and T. pseudokoningii. The second subgroup harbored T. simmonsii and T. lentiforme. Also, no correlation between genetic and geographic distance was detected. Conclusion The results of the present study indicated that the Saudi Arabian isolates showed greater nucleotide diversity compared to Egyptian isolates. Also, these findings will assist in future studies while assessing Trichoderma genetic diversity.