Reef fishes are critical functional groups for coral reefs, where the diversity of the former varies across different spatial scales. Multi-scale approaches are necessary to find this scale-dependence because the identification of such critical scales is fundamental to conserve coral reef biodiversity. For the first time, we present a study on fish diversity partition from the northern sector of the Mesoamerican Barrier Reef System (nsMBRS). In this area, coral reefs mainly present four habitats (e.g., lagoon, front, slope and terrace) and the spatial and environmental variables differ along a north-south gradient along the coast. This particular geomorphology provides an excellent opportunity to evaluate the reef fish diversity. Our objectives were to assess the inventory and differentiation diversity at habitat, reef and regional scales, and carry out additive diversity partitioning from sample to region scale. Total fish diversity (ε) was partitioned into its additive diversity components (αs, β_s, α, β, γ and δ), which were evaluated using bootstrap and rarefaction procedures, non-parametric statistics, and non-linear and null models. We found that α diversity was higher in the habitats front, slope and terrace, and β_h diversity was highest between lagoons and fronts. The most developed reefs exhibited the highest α, β_h, γ and δ_r diversity. The β_s and δ_r diversity were essentials to keep γ and ε diversity. Additive partition outcomes showed that total fish diversity is determined mostly by reef scale followed per sample and habitat scales. This supports the hypothesis that inter-habitat and reef differences seem to strongly regulate local and regional species richness. We conclude that reef scale was the most important level for conserving and keeping the biodiversity at nsMBRS.