Simple Summary: Deformed wing virus (DWV) is the most widespread bee virus and one of the most destructive pathogens affecting honeybees. Initially believed to be a honeybee virus with one dominant variant, DWV-A, we now know there are four master variants DWV-A, B (VDV-1), C and D. Since 2010, a shift in prevalence from the originally dominant DWV-A strain towards an increase in DWV-B prevalence has been observed in several countries. The geographical isolation of the bee populations in Hawaii provides a unique opportunity to examine the changes in DWV strain prevalence both at the individual colony and the apiary level. In this study, we present longitudinal data on the prevalence of DWV strains in individual colonies on the Island of Oahu, Hawaii. We found that although DWV-A was the dominant strain in the apiary, individual colonies could now be characterized as a mix of DWV-A and DWV-B and some tested positive for DWV-B only. Finally, DWV-B exhibited seasonal variations in both viral loads and prevalence in the apiary. Our long term DWV monitoring at the individual colony level contributes a unique perspective on the emerging patterns of DWV-B. The most prevalent viral pathogen of honeybees is Deformed Wing Virus (DWV) and its two most widely studied and common master-variants are DWV-A and DWV-B. The prevalence of DWV variants in the UK and in the US is changing, with the prevalence of the DWV-A strain declining and DWV-B increasing over time. In 2012, only DWV-A was detected on the Hawaiian Islands of Oahu. In this study we focused on a colony-level survey of DWV strains in a single apiary and examined the prevalence of DWV variants over the course of two years. In 2018 and 2019, a total of 16 colonies underwent viral testing in January, May, and September. Of those 16 colonies, four were monitored in both 2018 and 2019. Individual colonies showed variability of DWV master variants throughout the sampling period. DWV-A was consistently detected; however, the detection of DWV-B was variable across time in individual colonies. Ultimately, this study demonstrated a seasonal variation in both viral prevalence and load for DWV-B, providing a perspective on the dynamic nature of DWV master variants emerging in Hawaii. [ABSTRACT FROM AUTHOR]