In the present investigation PCR assays were developed for the rapid detection and differentiation of two poultry flagellates: Histomonas meleagridis and Tetratrichomonas gallinarum as well as the protozoan microorganism: Blastocystis spp. The nucleotide sequences of the small subunit ribosomal RNAs were used for primer construction obtaining fragments which vary in size for each microorganism. The established PCRs were able to detect DNA obtained from one microorganism of T. gallinarum and Blastocystis spp. propagated in vitro, proving the high analytical sensitivity of the method. DNA isolated from 10 protozoa was sufficient to detect H. meleagridis. To assess specificity, each PCR assay was performed with DNA from either H. meleagridis and/or T. gallinarum and/or Blastocystis spp. as well as with DNA from several other protozoan parasites (Eimeria tenella, Toxoplasma gondii, Cryptosporidia spp., Trichomonas gallinae, Entamoeba invadens, Entamoeba ranarum), fungi (Aspergillus fumigatus, Candida albicans), bacteria (Staphylococcae, Streptococcae, E. coli, Clostridium perfringens, Camplyobacter jejuni, Proteus) and viruses (fowl adenovirus serotype 4, avian reovirus) as well as livers and caecal samples from turkeys and specified pathogen free (spf) chickens. No cross-reactions with any of these samples were observed with the primer sets for the detection of H. meleagridis and Blastocystis spp. The primers designed for the identification of T. gallinarum yielded a PCR product with DNA of Trichomonas gallinae that had the identical size as the amplicon obtained with DNA from T. gallinarum. However, no PCR products resulted from any of the other samples tested with these primers. Liver and caecal samples from turkeys and chickens from flocks with outbreaks of histomonosis also named as "histomoniasis" originating from geographically distinct regions were investigated with the established PCRs. This is also the first report about the detection of the nucleic acid of H. meleagridis, T. gallinarum and Blastocystis spp. nucleic acid in the livers and/or caeca of laying hens and turkeys obtained from field outbreaks. Hence, the established PCR assays proved to be a rapid and sensitive diagnostic tool for the direct detection and differentiation of H. meleagridis, T. gallinarum and Blastocystis spp. nucleic acid in organ samples of infected turkeys and chickens regardless of the geographic origin.