The diversity of eukaryotic populations, in particular protozoa, in the water supplies of intensively reared broilers has not been previously studied. This important food-rearing environment was screened for the molecular diversity of eukaryotes by the analysis of PCR-amplified 18S rRNA. DNA was extracted from filtered water samples that were collected from the poultry drinking water systems of five farms. The total genomic DNA was used to produce rRNA-PCR amplicons, which, with the application of TTGE, provided an overview of the eukaryotic population diversity. The rRNA-PCR amplicons were then used to generate 34 random clones that were subject to comparative sequence analysis. Twenty-five of the clones (73.5%) showed high similarity with yeasts and fungi (> 92%) and 9 clones demonstrated similarity (> 86%) with certain protozoan groups, including flagellates and alveolates. Further studies of the microbial diversity in the previously ignored niche of intensively reared poultry drinking water systems are required, along with subsequent in vitro co-culture assays of the detected protozoa and bacterial strains.