Abstract Sugar beet is an economically important crop and one of the major sources of sucrose. Beet necrotic yellow vein virus (BNYVV) and Beet severe curly top virus (BSCTV) are two widespread viruses in sugar beet that cause severe damage to its performance. Previously, we have successfully achieved resistance to BNVYY by introducing coat protein-based DNA sequence constructs inducing gene silencing into sugar beet. However, the RNA silencing-mediated resistance of plants to a specific virus can be potentially broken down by another one as a part of synergistic interactions. In this study, we assayed the efficiency of the induced resistance of transgenic events to BNYVV and BSCTV-Ir under single or mixed infections. All the plants inoculated with just BSCTV-Ir displayed curly-leaf symptoms. However, partial resistance was observed in S3 events based on mild symptoms and low PCR amplification of the BSCTV-Ir coat protein sequence. Similarly, partial resistance to BSCTV-Ir was detected in the same transgenic plants under co-infection conditions. Based on the presented data, resistance to BNYVV was stable in almost all the transgenic plants co-infected with BSCTV-Ir, except for one event (S3-229) that was broken down. Overall, it seems that the co-infection with BNYVV and BSCTV-Ir does not affect the resistance of transgenic plants to both viruses. These findings demonstrated that RNA silencing-based resistance to BNYVV introduced in transgenic sugar beets of this study is very efficient and is not easily weakened after co-infection with a heterologous virus.