The elongation factor-1 alpha (EF-1α) is a highly conserved ubiquitous protein that is involved in translation and is desirable for use in phylogenetic studies on Blastocystis, an enigmatic intestinal parasite with a contentious taxonomic position. In the present study, a PCR product (BEα) that codes for a major part of the coding region of the EF-1α protein was amplified. Genome walking experiments together with cloning were implemented to elucidate the 5′ and 3′ ends of the EF-1α gene of the human isolate, Blastocystis hominis C. The genomic organization and the potential transcription factor binding sites of the 5′ end of B. hominis C EF-1α were identified. A comparative study on the deduced amino acid sequences of BEα of 13 Blastocystis isolates from various hosts was done to evaluate the phylogenetic relationship among the speciesThe nucleotide sequence data reported in this paper are available in the GenBank database with the accession numbers as shown in Table 1.. A phylogenetic reconstruction analysis with other eukaryotic EF-1α sequences was carried out to trace the phylogenetic position of Blastocystis among eukaryotic organisms.