The 1,021,348 base pair genome sequence of the Acanthamoeba polyphaga moumouvirus, a new member of the Mimiviridae family infecting Acanthamoeba polyphaga, is reported. The moumouvirus represents a third lineage beside mimivirus and megavirus. Thereby, it is a new member of the recently proposed Megavirales order. This giant virus was isolated from a cooling tower water in southeastern France but is most closely related to Megavirus chiliensis, which was isolated from ocean water off the coast of Chile. The moumouvirus is predicted to encode 930 proteins, of which 879 have detectable homologs. Among these predicted proteins, for 702 the closest homolog was detected in Megavirus chiliensis, with the median amino acid sequence identity of 62%. The evolutionary affinity of moumouvirus and megavirus was further supported by phylogenetic tree analysis of conserved genes. The moumouvirus and megavirus genomes share near perfect orthologous gene collinearity in the central part of the genome, with the variations concentrated in the terminal regions. In addition, genomic comparisons of the Mimiviridae reveal substantial gene loss in the moumouvirus lineage. The majority of the remaining moumouvirus proteins are most similar to homologs from other Mimiviridae members, and for 27 genes the closest homolog was found in bacteria. Phylogenetic analysis of these genes supported gene acquisition from diverse bacteria after the separation of the moumouvirus and megavirus lineages. Comparative genome analysis of the three lineages of the Mimiviridae revealed significant mobility of Group I self-splicing introns, with the highest intron content observed in the moumouvirus genome.