The biodiversity of reef fish in the Florida Keys National Marine Sanctuary was evaluated in terms of abundance, biomass, evenness, species richness, Shannon diversity, Simpson diversity, and functional diversity, using observations collected from 1999 – 2016 by the Reef Visual Census program. To compare the different diversity indices, species richness, Shannon diversity, Simpson diversity, and functional diversity were converted into effective number of species. We examined the seven indices by level of protection and type of no-take marine zones and by three habitat strata. The study detected abundance, biomass, and diversity were significantly greater (except evenness) inside no-take marine zones compared to areas open to fishing. Smaller reserves had higher abundance, biomass, and richness values than larger reserves and areas open to fishing, but had moderately higher diversity values. This may be attributed to a few species with many individuals that are dominant inside and outside no-take marine zones. Surprisingly, none of the indices were significantly different (except for functional diversity) between the larger Ecological Reserve and areas open for consumption. This may be due to spillover effects. Furthermore, the no-take marine zones only explained a small proportion of total percent deviance in the indices. Habitat type had a greater influence on patterns in composition and diversity where high relief reef habitats had the greatest abundance, biomass, and diversity indices. Based on our results managers should prioritize preserving high relief reefs through a network of small reserves to enhance reef fish composition and biodiversity.