To identify virus/viruses associated with the health of overwintering honey bee colonies, we investigated the viral infection status of 48 Apis mellifera ligustica colonies in three apiaries before and after overwintering. In one of the apiaries, the viral infection status of eight colonies was constantly monitored during the whole overwintering period. The results revealed that Deformed wing virus (DWV), Black queen cell virus (BQCV) and Israeli acute paralysis virus (IAPV) were highly prevalent in these colonies during overwintering period. Compared with the seemingly healthy colonies, the dying colonies after overwintering harbored a significantly higher load of IAPV, with around 1 × 10⁹ copies of genome. In the apiary in which colony losses happened, the genome copy number of DWV in honey bee colonies decreased, while that of IAPV increased. These results suggest that IAPV is the virus most associated with affecting the health of overwintering Apis mellifera ligustica colonies.