Abstract: Mitosporic fungal entomopathogens of the genus Lecanicillium (Ascomycota; Hypocreales) are of particular interest as biological control agents for phloem-sucking plant pests including aphids. Bioprospection for these fungi in Argentina has given rise to a set of isolates from a wide range of hosts. Molecular taxonomic analysis using a Multilocus Sequence Typing approach has confirmed previous morphology based assignments to the genus Lecanicillium.For further research in Lecanicillium fungi and development of a bio-aphicide, a PCR-based diagnostic tool allowing the reliable and fast differentiation of strain CEP419 from the further Argentine isolates was highly solicited. Screening of this full set of fungi and additional reference strains from other geographic origins for the presence or absence of self-splicing group-I introns disrupting the 18S and 28S rRNA encoding genes at previously identified intron insertion hot-spots revealed a unique intron constellation for the strain of particular interest. In contrast to all other Lecanicillium isolates investigated, the rRNA genes of CEP419 were found to comprise at least two group-I introns each. These findings were exploited in the development a double identification assay for CEP419. Primer pairs hybridizing against the 18S and 28S rRNA intron sequences, respectively, were designed and used to amplify partial rRNA gene sequences in a strain specific manner. Each of both diagnostic PCR reactions alone unambiguously identified strain CEP419 across the full set of Lecanicillium isolates investigated. In conclusion, the feasibility of strain-specific identification based on group-I intron sequences has been demonstrated for a potential biocontrol strain of Lecanicillium.