ABSTRACT The soil application of hydrothermally treated penicillin V fermentation residue (PFR) is attractive but challenged, due to the concern of the resistance risk in soil related to residual antibiotics. In this study, a lab-scale incubation experiment was conducted to investigate the influence of penicillin V on antibiotic resistance genes (ARGs) in PFR-amended soil via qPCR. The introduced penicillin V in soil could not be persistent, and its degradation occurred mainly within 2 days. The higher number of soil ARGs was detected under 108 mg/kg of penicillin V than lower contents (≤54 mg/kg). Additionally, the relative abundance of ARGs was higher in soil spiked with penicillin V than that in blank soil, and the great increase in the relative abundance of soil ARGs occurred earlier under 108 mg/kg of penicillin V than lower contents. The horizontal gene transfer might contribute to the shift of ARGs in PFR-amended soil. The results indicated that the residual penicillin V could cause the proliferation of soil ARGs and should be completely removed by hydrothermal treatment before soil application. The results of this study provide a comprehensive understanding of the resistance risk posed by penicillin V during the application of hydrothermally pretreated PFR.