Tigernut (Cyperus esculentus L.) is a perennial herb that is native to Africa and the Mediterranean coast. Due to the shortage of edible vegetable oil and biodiesel in China, tigernut has been emerging as a promising oil crop for its wide adaptability and high oil production per unit. To promote the development and utilization of this special crop, EPSPS (5-enolpyruvylshikimate-3-phosphate synthase) genes, which encode a key enzyme targeted by herbicide glyphosate in the shikimate pathway for the synthesis of aromatic amino acids, were identified from tigernut as well as several representative monocots based on genome and transcriptome data available. Comparative analyses revealed that (a) tigernut harbors a single EPSPS gene (denoted CeEPSPS) with seven introns as observed in most plant species; (b) its complete coding sequence of 1584 bp was further isolated using RT-PCR; (c) among 514 amino acids encoded by CeEPSPS, the first 70 residues from the N-terminal were characterized as the chloroplast signal peptide, whereas the peptide from sites 77 to 508 belongs to the highly conserved EPSP_ synthase domain (under the Pfam accession number of PF00275); (d) compared with the EPSP_ synthase domain, the chloroplast signal peptide is relatively more variable; and (e) the maturation protein of CeEPSPS was predicted to possess the theoretical molecular weight (Mw) of 47.32 kDa, the isolectric point (pI) of 5.49, the grand average of hydropathicity (GRAVY) of 0.069, the aliphatic index (AI) of 93.76, and the instability index (II) of 31.73, which is similar to other reported EPSPSs and could be categorized as a type of hydrophilic, acidic, and stable proteins; (f) phylogenetic analysis using deduced EPSPS proteins supports that tigernut is a Cyperaceae plant within Poales. Further sequence alignment and genome resequencing analyses revealed that no target site resistance mutations to glyphosate were found in all 56 germplasms investigated in this study. Gene expression analysis using qRT-PCR showed that CeEPSPS was dominantly expressed in mature leaves and tubers, which were significantly higher than that in buds, young leaves, senescent leaves, and rhizomes. Additionally, a plant vector of over-expressing CeEPSPS was also constructed, which would lay a solid foundation for the following molecular breeding for glyphosate resistant in tigernut and other species.