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Identifying <i>Eimeria</i> spp. circulating in a poultry flock assists in designing vaccine preventive programs, as different species do not cross-protect. Because species differ in anticoccidial drug susceptibility, species identification can also be used to optimize anticoccidial medication. In the present study, we designed pan-<i>Eimeria</i>-specific primers for the 18S rDNA and the cytochrome oxidase I (COI) genes, and tested whether next-generation sequencing of their amplicons allowed reliable identification of <i>Eimeria</i> spp. in samples of isolated oocysts. For each gene, two sets of primers to be used in a nested PCR (nPCR) system were designed. <i>In silico</i> evaluation of the primers using published sequences showed that nucleotide sequence identities of the nested amplicons were less than 97% between most species, while only identities of 18S rDNA genes of <i>Eimeria necatrix</i> and <i>Eimeria tenella</i> and between the COI genes of <i>Eimeria mitis</i> and <i>Eimeria mivati</i> were higher than 97%. Three vaccines and five <i>Eimeria</i> samples from chickens in backyard flocks were investigated by nPCRs and by direct PCRs (dPCR) using the nested (inner) primers with genomic DNA as the template. Seventeen further <i>Eimeria</i> samples from chickens in backyard flocks and three <i>Eimeria</i> samples from commercial broiler flocks were investigated only by nPCR. Sequencing nPCR products tended to detect more species than sequencing dPCR products and sequencing 18S rDNA products tended to detect more species than sequencing COI products. Regarding the detected species, there was a clear difference between the commercial broiler flocks and the backyard flocks. <i>Eimeria acervulina</i>, <i>Eimeria maxima</i>, and <i>E. tenella</i>/<i>E. necatrix</i> were the only species detected in broiler flocks, while the population in the backyard flocks was more varied, with <i>Eimeria brunetti</i> and <i>E. mitis</i>/<i>E. mivati</i> and the previously described operational taxonomic unit Y being more prevalent. Several sequences having less than 97% identity with one of the sequences used for clustering were detected in samples from backyard flocks. In conclusion, next-generation amplicon sequencing can be a useful tool to determine which <i>Eimeria</i> spp. are circulating in chicken flocks.