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The objectives of the current study were 1) to compare sample preparation workflows and quantitative real-time polymerase chain reaction assays (qPCR) as currently used in veterinary diagnostic laboratories with a study protocol utilizing commercially available reagents for individual Tritrichomonas foetus testing, 2) to assess the accuracy of pooling cultured smegma samples followed by extraction and qPCR testing as used in the study laboratory, and 3) to assess the specificity of the currently used primers and probes by sequencing all positive and presumptive positive samples identified in the study laboratory in an attempt to capture any nucleotide variability between T. foetus isolates and to rule out false-positive results possibly due to Simplicimonas moskowitzi. Eight hundred three cultured smegma samples were collected from different regions of the United States with the collaboration of 5 veterinary testing laboratories. The samples were processed individually by the respective laboratories, and then sent to the study laboratory and retested using the study protocol. Comparison testing showed an overall agreement of 95.89% between the veterinary testing laboratories and the study laboratory. One hundred seventy-six positive or presumptive positive samples plus 625 negative qPCR samples were combined and retested using a pooling protocol. Pools consisted of 1 positive sample and 4 negative samples (1/5). These pools were processed using the same study laboratory protocols, and 96% of the positive samples were detected in these pools. Nested PCR followed by sequencing confirmed 175 of the 178 samples classified as positive or presumptive positive in the study laboratory as containing T. foetus–specific DNA.