ABSTRACT. Molecular phylogenetic analyses have recently shown that the unicellular amoeboid protist Capsaspora owczarzaki is unlikely to be a nucleariid or an ichthyosporean as previously described, but is more closely related to Metazoa, Choanoflagellata, and Ichthyosporea. However, the specific phylogenetic relationship of Capsaspora to other protist opisthokont lineages was poorly resolved. To test these earlier results we have expanded both the taxonomic sampling and the number of genes from opisthokont unicellular lineages. We have sequenced the protein-coding genes elongation factor 1-α (EF1-α) and heat shock protein 70 (Hsp70) from C. owczarzaki and the ichthyosporean Sphaeroforma arctica. Our maximum likelihood (ML) and Bayesian analyses of a concatenated alignment of EF1-α, Hsp70, and actin protein sequences with a better sampling of opisthokont-related protist lineages indicate that C. owczarzaki is not clearly allied with any of the unicellular opisthokonts, but represents an independent unicellular lineage closely related to animals and choanoflagellates. Moreover, we have found that the ichthyosporean S. arctica possesses an EF-like (EFL) gene copy instead of the canonical EF1-α, the first so far described in an ichthyosporean. A maximum likelihood phylogenetic analysis shows that the EF-like gene of S. arctica strongly groups with the EF-like genes from choanoflagellates. Finally, to begin characterizing the Capsaspora genome, we have performed pulsed-field gel electrophoresis (PFGE) analyses, which indicate that its genome has at least 12 chromosomes with a total genome size in the range of 22–25 Mb.