Phosphoenolpyruvate carboxylases (PEPCs) are encoded by a small multigenic family. In order to characterise this gene family in sugarcane, seven DNA fragments displaying a high homology with grass PEPC genes were isolated using polymerase chain reaction-based cloning. A phylogenetic study revealed the existence of four main PEPC gene lineages in grasses and particularly in sugarcane. Moreover, this analysis suggests that grass C4 PEPC has likely derived from a root pre-existing isoform in an ancestral species. Using the Northern-dot-blot method, we studied the expression of the four PEPC gene classes in sugarcane cv. R570. We confirmed that transcript accumulation of the C4 PEPC gene (ppc-C4) mainly occurs in the green leaves and is light-induced. We also showed that another member of this gene family (ppc-aR) is more highly transcribed in the roots. The constitutive expression for a previously characterised gene (ppc-aL2) was confirmed. Lastly, the transcript accumulation of the fourth PEPC gene class (ppc-aL1) was not revealed. Length polymorphism in non-coding regions for three PEPC gene lineages enabled us to develop sequence-tagged site PEPC markers in sugarcane. We analysed the segregation of PEPC fragments in self-pollinated progenies of cv. R570 and found co-segregating fragments for two PEPC gene lineages. This supports the hypothesis that diversification of the PEPC genes involved duplications, probably in tandem.