This experimental and cross-sectional study was conducted in Lalibela National Apiculture Museum apiary site and surrounding kebeles in Lasta district, Ethiopia with overall objective of determining existence and prevalence of honeybee pest, particularly Varroa mite and its level of infestation during the year 2020. For the cross-sectional survey out of the five bordering kebeles, three kebeles and 100 beekeeper farmers were randomly selected and interviewed using prepared questioners. A total of 100 colonies were randomly selected from the beekeeper farmer’s apiaries (38 traditional, 32 transitional and 30 frame hives) and all 57 colonies of the museum that are hived in different hives (3 traditional, 12 transitional and 42 frame hives) were diagnosed for varroa and other pests. This study included two main components: The cross-sectional study and seasonal monitoring of varroa mite. Laboratory diagnosis was conducted according to the OIE and BEEBOOK standard protocols. The cross-sectional study results have indicated that the major pests and predators were wax moths, ants, spiders, bee eating birds, honey badger, varroa mites, lizards, beetles and bee lice were the most prominent once in order of their importance. Regarding the experimental diagnosis, 157 colonies (57colonies of the museum and 100 colonies of the beekeeper farmers were examined for the presence of varroa mite within the two main seasons (during active and dry dearth season 157 same colonies observation were taken repeatedly).The laboratory diagnosis result confirmed that 137(87.26%) were found positive for varroa mite. The infestation rate of varroa mite was; 9.461±0.639 and 10.628±0.649 in the museum apiary and in case of beekeeper farmer’s backyard colony 7.639±0.292 and 6.808±0.296 mites per hundred bees in phoretic and reproductive phases were observed, respectively. Furthermore, out of the 137 colonies positive for varroa mite 60 (75%)of the colonies from the beekeepers backyard and 49 (86%)from the museum apiary were infested >5% infestation level which has corresponded to an economic thresholds level of western honeybees. The explanatory variables that fit the general linear model: types of management, hive types and season were associated risk factors for the prevalence of varroa mites as hypothesized. Seasonal monitoring of varroa positive colonies indicated that phoretic stage varroa infestation level reached its peak starting from January to May and its reproductive stage from August to November. Finally, it could be concluded that the occurrence of varroa mite pests were a potential threats to the Lalibela National Apiculture Museum apiary and the surrounding local honeybees which needs an urge to devise and implement an appropriate control and prevention measures.