Real-time polymerase chain reaction (qPCR) is increasingly being used for detection of bovine leukemia virus (BLV) proviral DNA. Nevertheless, quality control for validation and standardization of such tests are currently lacking. Therefore, the present study was initiated by three OIE reference laboratories and three collaborating laboratories to measure inter-laboratory variability of six, already developed and available, BLV qPCR assays. For that purpose, an international panel of 58 DNA samples reflecting the dynamic range of the majority of the assays was distributed to 6 testing centers. Based on qualitative results, the overall agreement amongst all six laboratories was moderate. However, significant variability in BLV proviral DNA copy number measurement was observed amongst different laboratories. Quantitative PCR assays, even when performed by experienced staff, can yield large variability in BLV proviral DNA copy number without harmonization. Further standardization of different factors (i.e. utilization of unified protocols and unique calibrators) should increase inter-laboratory agreement.