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The cyanobacterium  Trichodesmium  plays an essential role supporting ocean productivity by relieving nitrogen limitation via dinitrogen (N 2 ) fixation. The two common Trichodesmium clades,  T. erythraeum  and  T. thiebautii , are both observed in waters along the West Florida Shelf (WFS). We hypothesized that these taxa occupy distinct realized niches, where  T. thiebautii  is the more oceanic clade. Samples for DNA and water chemistry analyses were collected on three separate WFS expeditions (2015, 2018, and 2019) spanning multiple seasons; abundances of the single copy housekeeping gene  rnpB  from both clades were enumerated via quantitative PCR. We conducted a suite of statistical analyses to assess Trichodesmium  clade abundances in the context of the physicochemical data. We observed a consistent coastal vs. open ocean separation of the two clades:  T. erythraeum  was found in shallow waters where the concentrations of dissolved iron (dFe) and the groundwater tracer Ba were significantly higher, while  T. thiebautii  abundance was positively correlated with water column depth. The Loop Current intrusion in 2015 with entrained Missisippi River water brought higher dFe and elevated abundance of both clades offshore of the 50 m isobath, suggesting that both clades are subject to Fe limitation on the outer shelf. Whereas, previous work has observed that  T. thiebautii  is more abundant than  T. erythraeum  in open ocean surface waters, this is the first study to examine  Trichodesmium  niche differentiation in a coastal environment. Understanding the environmental niches of these two key taxa bears important implications for their contributions to global nitrogen and carbon cycling and their response to global climate change.