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Among the causes of bee colony death, viruses are among the number of pathogens that can contribute to the ill health of a colony. This study focuses on two of the several honeybee viruses, <i>Slow Bee Paralysis Virus-1</i> (SBPV-1) and <i>Deformed Wing Virus B</i> (DWV-B), both belonging to the Iflavirus genus. To date, there is limited information available on the presence of these viruses in Italy. For this research project (IZS LT 04/19 RC), funded by the Ministry of Health, the presence and positivity of several honeybee viruses were evaluated in Italy using molecular methods. Convenience sampling was used, and these samples were analyzed for the two viruses and/or other main honeybee viruses, using specific PCR protocols and Sanger sequencing when necessary. A statistical analysis was conducted to study the independence between the viruses. Our data demonstrate for the first time the presence of SBPV-1 in Italian territory with a rather low number in most of the regions investigated, except for Emilia-Romagna and Lazio where it was detected more frequently, while DWV-B was detected at a higher level in all the regions investigated.