Twenty two monosporic Beauveria bassiana isolates obtained from maize soil were identified by molecular techniques. Genomic DNA was amplified by PCR using the primers pair ITS1 and ITS4. These primers amplified for 550 bp fragment that includes part of the 18 S subunit, ITS1, 5.8 S subunit, ITS2, and 28 S subunit of the ribosomal DNA. The DNA was cloned with E coli JM 109, and then a new PCR was carried out using the M13F and M13R primers. After that, the DNA product was sequenced it was assessed by the NCBI BLAST program, resulting a homology of 99% with other species of B. bassiana in the GeneBank. Ten monosporic B. bassiana isolates were evaluated at 1.2 × 10⁶ conidia/ml against neonate larvae of fall armyworm S. frugiperda. The better isolate of B. bassiana code CIDDB02 with assessed number KP860298 had LC₅₀ of 2.3 × 10⁷ conidia/ml, with 100% of insect mortality 72 h after conidia inoculation. This study allowed us to identify fast and reliably one B. bassiana isolated with highest pathogenic activity against the fall armyworm.