| Summary: | <i>Nosema ceranae</i> is a highly prevalent pathogen of <i>Apis mellifera</i>, which is distributed worldwide. However, there may still exist isolated areas that remain free of <i>N. ceranae</i>. Herein, we used molecular tools to survey the Azores to detect <i>N. ceranae</i> and unravel its colonisation patterns. To that end, we sampled 474 colonies from eight islands in 2014/2015 and 91 from four islands in 2020. The findings revealed that <i>N. ceranae</i> was not only present but also the dominant species in the Azores. In 2014/2015, <i>N. apis</i> was rare and <i>N. ceranae</i> prevalence varied between 2.7% in São Jorge and 50.7% in Pico. In 2020, <i>N. ceranae</i> prevalence increased significantly (<i>p</i> < 0.001) in Terceira and São Jorge also showing higher infection levels. The spatiotemporal patterns suggest that <i>N. ceranae</i> colonised the archipelago recently, and it rapidly spread across other islands, where at least two independent introductions might have occurred. Flores and Santa Maria have escaped the <i>N. ceranae</i> invasion, and it is remarkable that Santa Maria is also free of <i>Varroa destructor</i>, which makes it one of the last places in Europe where the honey bee remains naive to these two major biotic stressors.
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