Four group I introns, designated Bb1, Bb2, Bb3 and Bb4, were identified in the entomopathogenic hyphomycete Beauveria bassiana. Sequence analyses of these introns verified that they were invariably inserted at specific target sequences after conserved positions Ec2563, Ec2449, Ec2066 and Ec1921 of the large nuclear subunit (LSU) rDNA 3′-end. Secondary structure modelling confirmed that Bb1 and Bb3 belonged to subgroup IE while Bb2 and Bb4 belonged to subgroup IC1. Intron presence, distribution and size-variation were studied in a population of 125 B. bassiana strains using site-specific primers. Nucleotide sequences and secondary structures were compared and showed considerable variations usually at P1, P6 and P9 helices, but concomitantly, high homology between members of the same site-specific group. Intron distribution studies revealed that few (7.2%) strains were intron-less, most contained one (28%), two (48%) or three (16%) introns, while only one strain contained all four introns. Bb4-like introns (Ec1921) were the most abundant (86.4%), whereas the other three introns were evenly represented (ca 30%) in the B. bassiana population. Analysis of intron genotype distribution indicated a tenuous dependence upon geographic origin or insect host species. Phylogenetic analysis of all B. bassiana LSU introns and their close relatives from other entomopathogenic fungi showed a strong correlation between specific insertion sites and intron subgroups, fully supported by corresponding clades, suggesting common ancestry of the site specific LSU introns.