To assess interlaboratory variability in qualitative and quantitative Epstein-Barr virus (EBV) viral load (VL) testing, we distributed a panel of samples to 28 laboratories in the USA, Canada and Europe who performed testing using commercially available reagents (n = 12) or laboratory-developed assays (n = 18). The panel included two negatives, seven constructed samples using Namalwa and Molt-3 cell lines diluted in plasma (1.30–5.30 log10 copies/mL) and three clinical plasma samples. Significant interlaboratory variation was observed for both actual (range 1.30–4.30 log10 copies/mL) and self-reported (range, 1.70–3.30 log10 copies/mL) lower limits of detection. The variation observed in reported results on individual samples ranged from 2.28 log10 (minimum) to 4.14 log10 (maximum). Variation was independent of dynamic range and use of commercial versus laboratory-developed assays. Overall, only 47.0% of all results fell within acceptable standards of variation: defined as the expected result ± 0.50 log10. Interlaboratory variability on replicate samples was significantly greater than intralaboratory variability (p < 0.0001). Kinetics of change in VL appears more relevant than absolute values and clinicians should understand the uncertainty associated with absolute VL values at their institutions. The creation of an international reference standard for EBV VL assay calibration would be an initial important step in quality improvement of this laboratory tool.