Tonsil oral scrubbing (TOSc) is a simple and practical method recently developed for detecting PRRSV RNA in sows. This study evaluated how pooling and freeze-thaw procedures affect PRRSV RT-rtPCR results in TOSc samples. To assess pooling effects, 22 PRRSV-positive samples were divided into three Ct categories and diluted with negative TOSc samples at various ratios before testing. To assess freeze-thaw effects, 90 TOSc samples were split into two equal aliquots: one tested immediately, and the other tested after one freeze-thaw cycle. Results showed that PRRSV RNA detection probability decreased and Ct values increased as dilution (pooling) ratios rose. Samples with Ct values > 35 had significant drops in probability of detection beginning at a 1:3 dilution (≤71%). In contrast, samples with Ct values <35 maintained a high probability of detection until 1:10 dilution (≥85%). The freeze-thawed samples showed significantly higher Ct values and numerically lower probability of detection compared to non-freeze-thawed ones. In conclusion, pooling and freeze-thaw negatively impact PRRSV RNA detection in TOSc samples. Pooling may be acceptable for samples with a Ct value < 35 for up to 1:10 dilution, but should be avoided when the Ct value > 35. Similarly, the freeze-thaw process should be minimized during TOSc sample handling.