Endocannabinoid system evolution was estimated by searching for functional orthologs in the genomes of twelve phylogenetically diverse organisms: Homo sapiens, Mus musculus, Takifugu rubripes, Ciona intestinalis, Caenorhabditis elegans, Drosophila melanogaster, Saccharomyces cerevisiae, Arabidopsis thaliana, Plasmodium falciparum, Tetrahymena thermophila, Archaeoglobus fulgidus, and Mycobacterium tuberculosis. Sequences similar to human endocannabinoid exon sequences were derived from filtered BLAST searches, and subjected to phylogenetic testing with ClustalX and tree building programs. Monophyletic clades that agreed with broader phylogenetic evidence (i.e., gene trees displaying topographical congruence with species trees) were considered orthologs. The capacity of orthologs to function as endocannabinoid proteins was predicted with pattern profilers (Pfam, Prosite, TMHMM, and pSORT), and by examining queried sequences for amino acid motifs known to serve critical roles in endocannabinoid protein function (obtained from a database of site-directed mutagenesis studies). This novel transfer of functional information onto gene trees enabled us to better predict the functional origins of the endocannabinoid system. Within this limited number of twelve organisms, the endocannabinoid genes exhibited heterogeneous evolutionary trajectories, with functional orthologs limited to mammals (TRPV1 and GPR55), or vertebrates (CB2 and DAGLbeta), or chordates (MAGL and COX2), or animals (DAGLalpha and CB1-like receptors), or opisthokonta (animals and fungi, NAPE-PLD), or eukaryotes (FAAH). Our methods identified fewer orthologs than did automated annotation systems, such as HomoloGene. Phylogenetic profiles, nonorthologous gene displacement, functional convergence, and coevolution are discussed.