Lateral gene transfer plays an important role in the evolution of life. Events of ancient gene transfer can transmit genetic novelties to descendent lineages and subsequently shape their genetic systems. We here present the analyses of the gene encoding tyrosyl-tRNA synthetase (tyrRS), which reveal two eukaryotic tyrRS lineages, one including the opisthokonts and the other the remaining eukaryotes. The different origins of tyrRS lineages between the opisthokonts and the remaining eukaryotes indicate a likely case of ancient lateral gene transfer of tyrRS from an archaeon to the opisthokonts, which lends further support for the monophyly of the latter group. Ancient paralogy followed by differential gene loss is an alternative, albeit less parsimonious explanation for the distribution of the two eukaryotic tyrRS types. In either case, the presence of a haloarchaeal tyrRS type in the opisthokonts marks this group as monophyletic. This finding also points to the potential utility of ancient gene transfer events as molecular markers for major organismal lineages.