Abstract Background The rapid spread of SARS-CoV-2 has created a shortage of supplies of reagents for its detection throughout the world, especially in Latin America. The pooling of samples consists of combining individual patient samples in a block and analyzing the group as a particular sample. This strategy has been shown to reduce the burden of laboratory material and logistical resources by up to 80%. Therefore, we aimed to evaluate the diagnostic performance of the pool of samples analyzed by RT-PCR to detect SARS-CoV-2. Methods A cross-sectional study of diagnostic tests was carried out. We individually evaluated 420 samples, and 42 clusters were formed, each one with ten samples. These clusters could contain 0, 1 or 2 positive samples to simulate a positivity of 0, 10 and 20%, respectively. RT-PCR analyzed the groups for the detection of SARS-CoV-2. The area under the ROC curve (AUC), the Youden index, the global and subgroup sensitivity and specificity were calculated according to their Ct values that were classified as high (H: ≤ 25), moderate (M: 26–30) and low (L: 31–35) concentration of viral RNA. Results From a total of 42 pools, 41 (97.6%) obtained the same result as the samples they contained (positive or negative). The AUC for pooling, Youden index, sensitivity, and specificity were 0.98 (95% CI, 0.95–1); 0.97 (95% CI, 0.90–1.03); 96.67% (95% CI; 88.58–100%) and 100% (95% CI; 95.83–100%) respectively. In the stratified analysis of the pools containing samples with Ct ≤ 25, the sensitivity was 100% (95% CI; 90–100%), while with the pools containing samples with Ct ≥ 31, the sensitivity was 80% (95% CI, 34.94–100%). Finally, a higher median was observed in the Ct of the clusters, with respect to the individual samples (p < 0.001). Conclusions The strategy of pooling nasopharyngeal swab samples for analysis by SARS-CoV-2 RT-PCR showed high diagnostic performance.