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Seagrasses create foundational habitats in coastal ecosystems. One contributing factor to their global decline is disease, primarily caused by parasites in the genus Labyrinthula. To explore the relationship between seagrass and Labyrinthula spp. diversity in coastal waters, we examined the diversity and microhabitat association of Labyrinthula spp. in 2 inlets on Florida's Atlantic Coast, the Indian River Lagoon (IRL) and Banana River. We used amplicon-based high throughput sequencing with 2 newly designed primers to amplify Labyrinthula spp. from 5 seagrass species, water, and sediments to determine their spatial distribution and microhabitat associations. The SSU primer set identified 12 Labyrinthula zero-radius operational taxonomic units (ZOTUs), corresponding to at least 8 putative species. The ITS1 primer set identified 2 ZOTUs, corresponding to at least 2 putative species. Based on our phylogenetic analyses, which include sequences from previous studies that assigned seagrass-related pathogenicity to Labyrinthula clades, all but one of the ZOTUs that we recovered with the SSU primers were from non-pathogenic species, while the 2 ZOTUs recovered with the ITS1 primers were from pathogenic species. Some of the ZOTUs were widespread across the sampling sites and microhabitats (e.g. SSU ZOTU_10), and most were present in more than one site. Our results demonstrate that targeted metabarcoding is a useful tool for examining the relationships between seagrass and Labyrinthula diversity in coastal waters.