Bifunctional catalase-peroxidases (KatGs) are heme oxidoreductases widely spread among bacteria, archaea and among lower eukaryotes. In fungi, two KatG groups with different localization have evolved, intracellular (KatG1) and extracellular (KatG2) proteins. Here, the cloning, expression analysis and subcellular localization of two novel katG1 genes from the soil fungi Chaetomium globosum and Chaetomium cochliodes are reported. Whereas, the metalloenzyme from Ch. globosum is expressed constitutively, Ch. cochliodes KatG1 reveals a slight increase in expression after induction of oxidative stress by cadmium ions and hydrogen peroxide. The intronless open reading frames of both Sordariomycetes katG1 genes as well as of almost all fungal katG1s possess two peroxisomal targeting signals (PTS1 and PTS2). Peroxisomal localization of intracellular eukaryotic catalase-peroxidases was verified by organelle separation and immunofluorescence microscopy. Co-localization with the peroxisomal enzyme 3-ketoacyl-CoA-thiolase was demonstrated for KatGs from Magnaporthe grisea, Chaetomium globosum and Chaetomium cochliodes. The physiological role of fungal catalase-peroxidases is discussed.