Diagnosis of turkey Eimeria infection by conventional parasitologic methods is challenging and, until now, no molecular tools existed that clearly distinguished the four widely recognized pathogenic species: Eimeria adenoeides, E. meleagrimitis, E. gallopavonis, and E. dispersa. In this study, the internal transcribed spacer region one (ITS-1) was amplified and sequenced from 23 conventionally characterized reference samples. Phylogenic analysis segregated samples into five distinct cluster groups. The ITS-1 region(s) within each cluster were of a particular length and shared from 96% to 100% identity, while amplified ITS-1 region(s) between clusters differed in length and shared only 10.6% to 49.7% sequence identity. In addition, we developed PCR primer sets as diagnostic tools capable of specifically identifying members of each of the five clusters.