Tropilaelaps mites (Laelapidae) are ectoparasites of honey bees native to Southeast Asia. They reproduce and feed within brood, causing colony weakness, mortality, and significant production losses. The European Union (EU) currently remains free of this regulated disease. However, global trade and climate change are facilitating the spread of Tropilaelaps beyond its native range, becoming an increasingly threat to EU beekeeping. Therefore, EU official laboratories must be prepared to reliably identify these mites and ensure early diagnosis. In 2022-2023, a Tropilaelaps collection campaign was organised in Thailand by the European Union Reference Laboratory (EURL) for Bee health in collaboration with Chiang Mai University. The study aimed to: i) describe infestation patterns in managed Apis mellifera and wild Apis dorsata colonies, and ii) characterise Tropilaelaps specimens using morphological and molecular approaches. A total 80 A. mellifera brood samples (from 8 colonies across 10 apiaries) and 11 A. dorsata brood samples (from 11 colonies) were collected in northern Thailand. Mites (Tropilaelaps spp. and Varroa spp.) were extracted using standard procedures recommended by the World Organisation for Animal Health (WOAH) Tropilaelaps specimens were selected from two colonies per A. mellifera apiary and from three A. dorsata colonies. Morphological identification followed the validated EURL method, also described in the WOAH Terrestrial Manual. Genus confirmation was performed by COI conventional PCR, using EURL and WOAH protocols, and species identification by real-time PCR with high-resolution melting (HRM) analysis. Conventional PCR amplicons were sequenced using the same primers, and phylogenetic analyses were conducted on the resulting 538 bp fragments. Haplotypes were determined by comparison with reference sequences from international databases. In A. mellifera apiaries, Tropilaelaps were detected in 98% (78/80) of sampled colonies, with apiary-level prevalence ranging from 88 to 100%. Co-infestation with Varroa spp. occurred in 70% (7/10) of apiaries and 35% (28/80) of colonies, with a prevalence of co-infested colonies within apiaries ranging from 0% to 88%. Tropilaelaps mercedesae was the only species identified in both A. mellifera (European honey bee) and A. dorsata colonies (giant honey bee, original host of T. mercedesae). Phylogenetic analyses showed that some sequences were closely related to sequences from Thailand, with no clear genetic differentiation between the two host species. These results provide well-characterised reference material for EU official laboratories and valuable epidemiological data to support surveillance and risk mitigation strategies against Tropilaelaps introduction into the EU.