BACKGROUND: Caleosin/peroxygenases, CLO/PXG, (designated PF05042 in Pfam) are a group of genes/proteins with anomalous distributions in eukaryotic taxa. We have previously characterised CLO/PXGs in the Viridiplantae. The aim of this study was to investigate the evolution and functions of the CLO/PXGs in the Fungi and other non-plant clades and to elucidate the overall origin of this gene family. RESULTS: CLO/PXG-like genes are distributed across the full range of fungal groups from the basal clades, Cryptomycota and Microsporidia, to the largest and most complex Dikarya species. However, the genes were only present in 243 out of 844 analysed fungal genomes. CLO/PXG-like genes have been retained in many pathogenic or parasitic fungi that have undergone considerable genomic and structural simplification, indicating that they have important functions in these species. Structural and functional analyses demonstrate that CLO/PXGs are multifunctional proteins closely related to similar proteins found in all major taxa of the Chlorophyte Division of the Viridiplantae. Transcriptome and physiological data show that fungal CLO/PXG-like genes have complex patterns of developmental and tissue-specific expression and are upregulated in response to a range of biotic and abiotic stresses as well as participating in key metabolic and developmental processes such as lipid metabolism, signalling, reproduction and pathogenesis. Biochemical data also reveal that the Aspergillus flavus CLO/PXG has specific functions in sporulation and aflatoxin production as well as playing roles in lipid droplet function. CONCLUSIONS: In contrast to plants, CLO/PXGs only occur in about 30% of sequenced fungal genomes but are present in all major taxa. Fungal CLO/PXGs have similar but not identical roles to those in plants, including stress-related oxylipin signalling, lipid metabolism, reproduction and pathogenesis. While the presence of CLO/PXG orthologs in all plant genomes sequenced to date would suggest that they have core housekeeping functions in plants, the selective loss of CLO/PXGs in many fungal genomes suggests more restricted functions in fungi as accessory genes useful in particular environments or niches. We suggest an ancient origin of CLO/PXG-like genes in the 'last eukaryotic common ancestor' (LECA) and their subsequent loss in ancestors of the Metazoa, after the latter had diverged from the ancestral fungal lineage.