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Trypanosomatids form a group of high prevalence protozoa that parasitise honey bees, with Lotmaria passim as the predominant species worldwide. However, the knowledge about the ecology of trypanosomatids in isolated areas is limited. The Portuguese archipelagos of Madeira and Azores provide an interesting setting to investigate these parasites because of their geographic isolation, and because they harbour honey bee populations devoid of two major enemies: Varroa destructor and Nosema ceranae. Hence, a total of 661 honey bee colonies from Madeira and the Azores were analysed using different molecular techniques, through which we found a high prevalence of trypanosomatids despite the isolation of these islands. L. passim was the predominant species and, in most colonies, was the only one found, even on islands free of V. destructor and/or N. ceranae with severe restrictions on colony movements to prevent the spread of them. However, islands with V. destructor had a significantly higher prevalence of L. passim and, conversely, islands with N. ceranae did not shown any significant correlation with the trypanosomatid. Crithidia bombi was detected in Madeira and on three islands of the Azores, almost always coincident with L. passim. By contrast, Crithidia mellificae was not detected in any sample. A high-throughput sequencing analysis distinguished two main haplotypes of L. passim, which accounted for 98% of the total sequence reads. This work suggests that L. passim and C. bombi are parasites that have been associated with honey bees predating the spread of V. destructor and N. ceranae., (© 2023. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)