The plant and microbial peroxidase superfamily encompasses three classes of related protein families. Class I includes intracellular peroxidases of prokaryotic origin, class II includes secretory fungal peroxidases, including the lignin degrading enzymes manganese peroxidase (MnP), lignin peroxidase (LiP), and versatile peroxidase (VP), and class III includes the secretory plant peroxidases. Here, we present phylogenetic analyses using maximum parsimony and Bayesian methods that address the origin and diversification of class II peroxidases. Higher-level analyses used published full-length sequences from all members of the plant and microbial peroxidase superfamily, while lower-level analyses used class II sequences only, including 43 new sequences generated from Agaricomycetes (mushroom-forming fungi and relatives). The distribution of confirmed and proposed catalytic sites for manganese and aromatic compounds in class II peroxidases, including residues supposedly involved in three different long range electron transfer pathways, was interpreted in the context of phylogenies from the lower-level analyses. The higher-level analyses suggest that class II sequences constitute a monophyletic gene family within the plant and microbial peroxidase superfamily, and that they have diversified extensively in the basidiomycetes. Peroxidases of unknown function from the ascomycete Magnaporthe grisea were found to be the closest relatives of class II sequences and were selected to root class II sequences in the lower-level analyses. LiPs evidently arose only once in the Polyporales, which harbors many white-rot taxa, whereas MnPs and VPs are more widespread and may have multiple origins. Our study includes the first reports of partial sequences for MnPs in the Hymenochaetales and Corticiales.