Chicken coccidia are protozoan parasites of the genus Eimeria. They cause economical losses in the poultry industry globally. The various species can be distinguished on the basis of the morphology of the oocysts and parasitic site in intestine, but these criteria sometimes are unreliable. Therefore, a species-specific polymerase chain reaction (PCR) was developed. Based on variable sequence regions, specific primers were constructed for the differentiation of five Eimeria species (Eimeria acervulina, E. brunette, E. maxima, E. necatrix, and E. tenella). PCR products were amplified from coccidian vaccine (coccivac-D and coccivac-B) and E. tenella and were subsequently sequenced. Similarities of the five species sequences between the vaccines and Genbank were 94-100%. Analysis of the E. tenella internal transcribed spacer 1 (ITS-1) partial sequence from Taiwan and from Genbank indicated that the similarity was 99.6%. The PCR sensitivity test of E. tenella in Taiwan is 50 oocysts. The five sets of primers will not amplify any non-specific bands of the chicken genome or its intestinal contents. Therefore, the five sets of specifically designed primers are guaranteed to be useful for differential diagnosis of avian coccidiosis caused by Eimeria spp.