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Background and Aim: Tilapia aquaculture is rapidly expanding across Southeast Asia and plays a critical role in regional food security. While bacterial microbiomes of farmed fish have been widely investigated, the fungal component of aquatic microbial communities remains poorly characterized, particularly at the biologically important interface between rearing water and fish gills. Fungi may influence fish health, environmental microbial ecology, and occupational exposure risks within aquaculture systems. This study aimed to characterize fungal mycobiomes associated with rearing water and gills of clinically healthy red tilapia (Oreochromis spp. hybrids) cultured in Central Thailand using internal transcribed spacer (ITS) rRNA amplicon sequencing and to determine how habitat type, farming system, and environmental variables shape fungal community structure. Materials and Methods: Samples were collected from ten tilapia farms located in five provinces of Central Thailand, representing two aquaculture systems: open river cages and closed earthen ponds. A total of 27 rearing water samples and 30 composite gill samples were analyzed. Fungal DNA was extracted and the ITS1 region was amplified and sequenced using the Illumina MiSeq platform. Sequence processing and amplicon sequence variant inference were performed in QIIME2 using the DADA2 pipeline. Alpha diversity indices and beta diversity analyses were used to evaluate community structure, while multivariate statistical approaches assessed the influence of habitat type, geographic location, farming style, and physicochemical water parameters. Results: Fungal communities displayed considerable taxonomic diversity and differed significantly between habitats. Rearing water samples exhibited significantly higher alpha diversity than gill-associated communities. Dominant genera included Cladosporium, Candida, Aspergillus, Fusarium, and Rhodotorula. Gill communities were relatively enriched in Candida and Fusarium, whereas rearing water contained higher abundances of Cladosporium and Rhodotorula. Beta diversity analyses demonstrated significant effects of sampling source, province, and farming system on fungal community composition. Environmental parameters such as pH, nitrate concentration, and ionic strength were associated with variations in fungal diversity, particularly in rearing water. Several detected genera included taxa with known opportunistic pathogenic potential for fish and humans. Conclusion: This study provides the first ITS-based baseline characterization of fungal mycobiomes associated with red tilapia aquaculture systems in Central Thailand. Distinct fungal assemblages occur at the water–gill interface, with environmental conditions and aquaculture practices influencing community composition. The presence of opportunistic fungal genera highlights the importance of incorporating fungal community monitoring into aquaculture biosecurity and One Health surveillance frameworks to support sustainable fish production, environmental health, and occupational safety. Keywords: aquaculture microbiome, fish gills, fungal diversity, fungal mycobiome, ITS rRNA sequencing, One Health, red tilapia, water microbiome.