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Purpureocillium lilacinum has been widely used as a commercial biocontrol agent for the control of plant parasitic nematodes. Whole genome analysis promotes the identification of functional genes and the exploration of their molecular mechanisms. The Cyclophilin (CYP) gene family belongs to the immunophillin superfamily, and has a conserved cyclophilin-like domain (CLD). CYPs are widely identified in prokaryotes and eukaryotes, and can be divided into single- and multi-domain proteins. In the present study, 10 CYP genes possessing the CLD, named PlCYP1–P10, were identified from the genome of P. lilacinum strain 36-1. Those 10 PlCYPs were predicted to have different cellular localizations in P. lilacinum. Phylogenetic and gene structure analysis revealed the evolutionary differentiation of CYPs between Ascomycotina and Saccharomycotina fungi, but conservation within the Ascomycotina fungi. Motif and gene structure distributions further support the result of phylogenetic analysis. Each PlCYP gene had a specific expression pattern in different development stages of P. lilacinum and its parasitism stage on eggs of Meloidogyne incognita. In addition, the 10 PlCYP genes exhibited different expression abundances in response to abiotic stresses, among which PlCYP4 was highly expressed at a high temperature (35 °C), while PlCYP6 was up-regulated under 5 mM of H2O2 stress. Furthermore, the heterologous expression of PlCYP4 and PlCYP6 in Escherichia coli enhanced the cellular tolerance against a high temperature and H2O2. In summary, our study indicates the potential functions of PlCYPs in virulence and the stress response, and also provides a frame for further analysis of the CYP gene family in Ascomycotina fungi. [ABSTRACT FROM AUTHOR]