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Abstract Thirteen fungal isolates, having characteristics similar to Beauveria bassiana, were isolated from field soils of Punjab, India, on Beauveria-specific selective medium (BS medium). Molecular screening of the B. bassiana isolates, using PCR amplification with B. bassiana-specific primer and nucleotide sequence analysis of ITS region, confirmed only three isolates as B. bassiana (now named as BbR1, BbR2, and BbR3) along with MTCC 2028 reference isolate. Comparative RAPD-PCR amplification with ten different RAPD primers showed that all the three isolates were closely related. Genetic relatedness dendrogram developed, using RAPD-PCR data by UPGMA, measured the quantitative description of genetic diversity and showed that the standard isolate MTCC2028 was 58% similar to new isolates. B. bassiana isolates achieved their maximum growth during the optimum incubation period of 7 days. Significant mortality rates of the tobacco caterpillar, Spodoptera litura (Fabricius) (Lepidoptera: Noctuidae), were recorded at all the tested concentrations of conidial suspensions (103 to 107 conidia ml−1) of B. bassiana. BbR2 was found to be the most potential local isolate, causing the maximum mortality (83.33%) of S. litura, with LC50 value of 935.663 conidia ml−1, followed by BbR3 (74.33%), BbR1 (72.22%), and BbM1 (67.12%) isolates.