A denaturing gradient gel electrophoresis (DGGE) assay was developed to rapidly discriminate species of avian Eimeria. Amplification by PCR of the small subunit ribosomal RNA gene (approximately 1,600 nucleotides) with Eimeria genus-specific primers followed by cloning and sequencing allowed us to carry out phylogenetic analyses and identify clone sequences to species level in most cases. Clones were subsequently used to amplify a smaller fragment (approximately 120 nucleotides) suitable for DGGE. The fragments were separated on denaturing gradient gel and bands with unique migration distances were mixed to obtain an identification ladder. The identification ladder and PCR products obtained from DNA extracted from fecal samples from several poultry farms were compared. Applying the DGGE method in this study allowed a rapid differentiation of Eimeria species present in fecal samples collected from poultry farms.