Background and Aims Rapid and accurate detection of COVID-19 is crucial for mitigation of the pandemic. We evaluated the performance of six molecular kits and the effect of several factors on the performance of the kits. Materials and Methods Two hundred and four nasopharyngeal samples were collected from participants aged ≥18 years at the Baruch Padeh Medical Center Poriya, Israel, between June and August 2020. Samples were tested by: Allplex 2019‐nCOV Assay (Seegene), Real‐Time Fluorescent RT‐PCR Kit for Detecting SARS-2019-nCoV (BGI Genomics), Xpert ® Xpress SARS-CoV-2 test (Cepheid), Simplexa ® COVID-19 Direct Kit (Focus Diagnostics), BD SARS-CoV-2 Reagents for BD MAX™ System (BD), and Logix Smart™ Coronavirus Disease 2019 (COVID-19) Test kit (CO-DIAGNOSTICS). Results Xpert ® Xpress SARS-CoV-2 test and Logix Smart™ COVID-19 Kit had the highest (91.2%) and the lowest (74.5%) sensitivity, respectively. Symptoms were a predictor of a positive result. Traditional assays had a higher minimum cycle threshold (min Ct), i.e. detected lower viral load, compared to rapid assays ( p = 0.012). Samples of symptomatic participants had lower min Ct, than samples of asymptomatic participants ( p < 0.001). Additionally, the more genes were detected, the lower the min Ct ( p < 0.001), indicating that a greater percentage of the viral genome was amplified. Conclusions Taken together, most assays had overall good performance. Since several factors affect the performance of kits, each laboratory must be familiar with its kit’s limitations in order to produce the most reliable results. Supplementary Information The online version contains supplementary material available at 10.1007/s40291-021-00574-y.