Most of the major groups of eukaryotes have microbial representatives that thrive in low oxygen conditions. Those that have been studied in detail generate ATP via pathways involving anaerobically functioning enzymes of pyruvate catabolism that are typically absent in aerobic eukaryotes and whose origins remain controversial. These enzymes include pyruvate:ferredoxin oxidoreductase, pyruvate:NADP(+) oxidoreductase, and pyruvate formate lyase (Pfl). Pfl catalyzes the nonoxidative generation of formate and acetyl-Coenzyme A (CoA) from pyruvate and CoA and is activated by Pfl activating enzyme (Pfla). Within eukaryotes, this extremely oxygen-sensitive pathway was first described in the hydrogenosomes of anaerobic chytrid fungi and has more recently been characterized in the mitochondria and chloroplasts of the chlorophyte alga Chlamydomonas reinhardtii. To clarify the origins of this pathway, we have comprehensively searched for homologs of Pfl and Pfla in publicly available large-scale eukaryotic genomic and cDNA sequencing data, including our own from the anaerobic amoebozoan Mastigamoeba balamuthi. Surprisingly, we find that these enzymes are widely distributed and are present in diverse facultative or obligate anaerobic eukaryotic representatives of the archaeplastidan, metazoan, amoebozoan, and haptophyte lineages. Using maximum likelihood and Bayesian phylogenetic methods, we show that the eukaryotic Pfl and Pfla sequences each form monophyletic groups that are most closely related to homologs in firmicute gram-positive bacteria. Topology tests exclude both α-proteobacterial and cyanobacterial affinities for these genes suggesting that neither originated from the endosymbiotic ancestors of mitochondria or chloroplasts. Furthermore, the topologies of the eukaryote portion of the Pfl and Pfla trees significantly differ from well-accepted eukaryote relationships. Collectively, these results indicate that the Pfl pathway was first acquired by lateral gene transfer into a eukaryotic lineage most probably from a firmicute bacterial lineage and that it has since been spread across diverse eukaryotic groups by more recent eukaryote-to-eukaryote transfer events.