Gobies are the most diverse marine fish family. Here, we analysed the gamma-diversity (γ-diversity) partitioning of gobiid fishes to evaluate the additive and multiplicative components of α and β-diversity, species replacement and species loss and gain, at four spatial scales: sample units, ecoregions, provinces and realms. The richness of gobies from the realm Eastern Tropical Pacific (ETP) is represented by 87 species. Along latitudinal and longitudinal gradients, we found that the γ-diversity is explained by the β-diversity at both spatial scales, ecoregions and provinces. At the ecoregion scale, species are diverse in the north (Cortezian ecoregion) and south (Panama Bight ecoregion) and between insular and coastal ecoregions. At the province scale, we found that the species turnover between the warm temperate Northeast Pacific (WTNP), Tropical East Pacific (TEaP) and the Galapagos Islands (Gala) was high, and the species nestedness was low. At the ecoregion scale, historical factors, and phylogenetic factors have influenced the hotspots of gobiid fish biodiversity, particularly in the Cortezian, Panama Bight and Cocos Island ecoregions, where species turnover is high across both latitudinal and longitudinal gradients. At the provincial level, we found that the contributions of the β-diversity from north to south, in the WTNP, TEaP and Gala were high, as result of the high number of unique species. Species turnover was also high at this scale, with a low contribution from species nestedness that was probably due to the low species/gene flow within the provinces. These results highlight the importance and successful inclusion of a cryptobenthic fish component in ecological and biogeographical studies.