RNA viruses that affect honeybees have been involved in colony losses reported around the world. The aim of the present work was to evaluate the prevalence and distribution of honeybee viruses during 2006–2007 in Spanish professional apiaries, and their association with colony losses. Four hundred and fifty-six samples from apiaries located in different geographic regions of Spain were analyzed. Thirty-seven percent of the samples had viral presence. Most (80%) had one virus and 20% two different viruses. All the analyzed viruses, Deformed Wing Virus (DWV), Israeli Acute Paralysis Virus (IAPV), Black Queen Cell Virus (BQCV), Sacbrood Virus (SBV) and Kashmir Bee Virus (KBV) were detected, but detection rates were lower than expected. According to these results and considering the high prevalence of other honeybee pathogens in Spain, the role of viruses in colony losses in Spain may be discussed.