Varroa destructor is a major pest in world beekeeping. It was first detected in Madagascar in 2010 on the endemic honeybee Apis mellifera unicolor. To evaluate V. destructor spread dynamics in Madagascar a global survey was conducted in 2011–2012. A total of 695 colonies from 30 districts were inspected for the presence of mites. 2 years after its introduction, nine districts were found infested. Varroa destructor spread was relatively slow compared to other countries with a maximum progression of 40 km per year, the five newly infested districts being located next to the first infested ones. The incidence of mite infestation was also investigated by monitoring 73 colonies from five apiaries during 1 year (2011–2012). Sixty percent of local colony mortality was recorded after 1 year of survey. Varroa destructor strain determination was done by partial sequencing of the cytochrome oxidase I gene of 13 phoretic mites sampled in five districts. A single V. destructor mitochondrial haplotype was detected, the Korean type, also present in the closest African countries. A global pathogen survey was also conducted on the colonies inspected for mite presence. The greater wax moth, Galleria mellonella has been found in all colonies all over the country. Two other pathogens and morphological abnormalities in workers, such as deformed wings, were found associated with only V. destructor presence. A prevention management plan must be implemented to delimit mite spread across the island.