AIMS: Agrobacterium tumefaciens-mediated transformation (AtMT) is an effective method for generation of entomopathogenic Beauveria bassiana transformants. However, some strains grow on the selective medium containing hygromycin B (HygB), which reduces the selection efficiency of the putative transformants. In this work, a relationship between HygB resistance gene promoter and AtMT efficiency was investigated to improve the transformant selection. METHODS AND RESULTS: Ten B. bassiana isolates were grown on 800 μg ml-1 HygB medium, but only JEF-006, -007 and -013 showed susceptibility to the antibiotics. Particularly, JEF-007 showed the most dose-dependent susceptibility. Two different Ti-Plasmids, pCeg (gpdA promoter based) and pCambia-egfp (CaMV 35S promoter based), were constructed to evaluate the promoters on the expression of HygB resistance gene (hph) at 100, 150 and 200 μg ml-1 HygB medium. Eight days after the transformation, wild type, AtMT/pCeg and AtMT/pCambia-egfp colonies were observed on 100 μg ml-1 HygB, but significantly larger numbers of colonies were counted on AtMT/pCeg plates. At higher HygB concentration (150 μg ml-1 ), only AtMT/pCeg colonies were further observed, but very few colonies were observed on the wild type and AtMT/pCambia-egfp plates. Putative transformants were subjected to PCR, RT-PCR and qRT-PCR to investigate the T-DNA insertion rate and gene expression level. Consequently, >80% of colonies showed successful AtMT transformation, and the hph expression level in AtMT/pCeg colonies was higher than that of AtMT/pCambia-egfp colonies. CONCLUSIONS: In the HygB-susceptible B. bassianaJEF-007, gpdA promoter works better than CaMV 35S promoter in the expression of HygB resistance gene at 150 μg ml-1 HygB, consequently improving the selection efficiency of putative transformants. SIGNIFICANCE AND IMPACT OF THE STUDY: These results provide useful information for determining AtMT effectiveness in B. bassiana isolates, particularly antibiotic susceptibility and the role of promoters.