In this study, the cytochrome b (Cyt b) amino acid sequences were analyzed in 50 organisms covering all 5 kingdoms of eukaryotes. Six conserved domains, i.e., heme bL binding sites, heme bH binding sites, Qo binding sites, Qi binding sites, the interchain domain interface, and the intrachain domain interface were found in all investigated sequences. The topology of the phylogenetic trees was largely consistent with the well recognized taxonomic relationships, indicating that the Cyt b genes originated from a common ancestral gene before the divergence of eukaryotic kingdoms. The eukaryotic Cyt b genes likely originated from an ancient prokaryotic gene in Alphaproteobacteria based on shared conserved domains. We provide evidence that the Cyt b gene of oomycete Pseudoperonospora cubensis was horizontally transferred from a fungus in the order Hypocreales. To our knowledge, this is the first reported evidence of Horizontal gene transfer (HGT) from Fungi to Chromista involving an essential house-keeping gene. Our data suggest that HGT events must be considered when evolutionary trees are constructed only based on Cyt b genes. Additional analysis of thousands of Cyt b sequences from Genbank revealed that introns in mitochondrial Cyt b genes were acquired after the endosymbiosis of alphaproteobacteria in eukaryotic cells.