Species that have complex courtship behaviors are some of the most evolutionarily diverse lineages observed in nature. Divergent, pre-mating calls are effective in both generating and maintaining reproductive isolation. Complex courtship displays provide numerous traits in which a small change can reinforce or lead to reproductive isolation. And display traits often evolve interactively, multiplicatively increasing the potential phenotype variants. Because many traits can be quantified and used to document variation among species, organisms that use complex courtship behaviors provide model systems for testing the influence of ecology on lineage diversification and trait evolution. Here, we quantify differences in the courtship behavior, morphology, and genetic trait change of male Photeros annecohenae over an intermediate range of geographic distances along reef habitats of the Mesoamerican barrier reef of Belize. Differences in bioluminescent ostracod behavior, morphology, and genetics have been documented across large geographic distances (500-1000 km) and at smaller geographic scales (~12km) but intermediate spatial scales have not previously been evaluated. We found significant differences in observed behavioral, morphological, and genetic traits across isolated populations of P. annecohenae resulting from both isolation by distance (IBD) and isolation by barrier (IBB). Furthermore, we describe a newly discovered downward displaying behavioral variant population of P. annecohenae nested within the upward displaying populations. The morphological, behavioral, and genetic variability documented for P. annecohenae across the 196 km seagrass mosaic of the Mesoamerican reef system offers novel insight toward our understanding of the speciation continuum and the role of complex behavioral courtship in promoting divergence within taxa.