Grapevine leafroll-associated virus 3 (GLRaV-3) is associated with grapevine leafroll disease, one of the most economically important viral diseases of grapevines. This disease impacts on both vine health and grape quality; reduction in yield, brix and wine colour are among its detrimental effects. Many methods, including serological and molecular procedures, have been developed for the detection of GLRaV-3; however, there is no PCR-based assay available to quantify virus populations within plant tissues. A real-time RT-PCR assay with TaqMan probe was developed for specific and reliable quantitative detection of GLRaV-3 in infected tissues. The designed primers and probes target the conserved sequence in the RNA-dependent RNA polymerase (RdRp) domain of the viral genome to prevent amplification of most subgenomic and defective RNAs. This protocol was used to examine the seasonal dynamics and translocation of GLRaV-3 in field-grown grapevines. The results showed that the virus spread quickly from trunks to new growing shoots and leaves early in the growing season, and most samples still harboured detectable virus during late summer and autumn. The seasonal progress of one GLRaV-3 isolate was compared in four grapevine cultivars (Chardonnay, Cabernet Sauvignon, Italia and Thompson Seedless). Within cultivars there was little variability in the distribution and translocation of GLRaV-3, except for in Thompson Seedless. This quantitative detection assay will be a valuable tool for GLRaV-3 diagnosis, disease monitoring and population ecology studies.