Objective To construct convenient CRISPR/Cas9-mediated genome editing systems in industrial enzyme-producing fungiPenicillium oxalicumandTrichoderma reesei. Results Employing the 5S rRNA promoter fromAspergillus nigerfor guide RNA expression, the beta-glucosidase genebgl2inP. oxalicumwas deleted using a donor DNA carrying 40-bp homology arms or a donor containing no selectable marker gene. Using a markerless donor DNA as editing template, precise replacement of a small region was achieved in thecreAgene. InT. reesei, theA. niger5S rRNA promoter was less efficient than that inP. oxalicumwhen used for gene editing. Using a native 5S rRNA promoter, stop codons were introduced into thelae1coding region using a markerless donor DNA with an editing efficiency of 36.67%. Conclusions Efficient genome editing systems were developed in filamentous fungiP. oxalicumandT. reeseiby using heterologous or native 5S rRNA promoters for guide RNA expression.