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Cotton leaf curl disease (CLCuD) is a significant constraint to the economies of Pakistan and India. The disease is caused by different begomoviruses (genus Begomovirus, family Geminiviridae) in association with a disease-specific betasatellite. However, another satellite-like molecule, alphasatellite, is occasionally found associated with this disease complex. A quantitative real-time PCR assay for the virus/satellite components causing CLCuD was used to investigate the performance of selected cotton varieties in the 2014–2015 National Coordinated Varietal Trials (NCVT) in Pakistan. The DNA levels of virus and satellites in cotton plants were determined for five cotton varieties across three geographic locations and compared with seed cotton yield (SCY) as a measure of the plant performance. The highest virus titer was detected in B-10 (0.972 ng·µg⁻¹) from Vehari and the lowest in B-3 (0.006 ng·µg⁻¹) from Faisalabad. Likewise, the highest alphasatellite titer was found in B-1 (0.055 ng·µg⁻¹) from Vehari and the lowest in B-1 and B-2 (0.001 ng·µg⁻¹) from Faisalabad. The highest betasatellite titer was found in B-23 (1.156 ng·µg⁻¹) from Faisalabad and the lowest in B-12 (0.072 ng·µg⁻¹) from Multan. Virus/satellite DNA levels, symptoms, and SCY were found to be highly variable between the varieties and between the locations. Nevertheless, statistical analysis of the results suggested that betasatellite DNA levels, rather than virus or alphasatellite DNA levels, were the important variable in plant performance, having an inverse relationship with SCY (−0.447). This quantitative assay will be useful in breeding programs for development of virus resistant plants and varietal trials, such as the NCVT, to select suitable varieties of cotton with mild (preferably no) symptoms and low (preferably no) virus/satellite. At present, no such molecular techniques are used in resistance breeding programs or varietal trials in Pakistan. Full article