The Guiana Shield region in northern South America has a rich and diverse ichthyofauna with a high degree of endemism. The fauna of this region is shaped not only by the geology of the area but climatic events as well. The Rupununi district of southwestern Guyana is an area where seasonal rainfall floods a vast savanna. During the rainy season, this inundated savanna allows a connection between the Amazon and Essequibo Rivers that provides the potential for ichthyofaunal exchange. The connection is referred to as the Rupununi portal and this study investigates how this feature influences fish distributions and diversity between the drainages it links. In this study, fishes on either side of the portal were extensively sampled. Statistical comparisons of fish community structure from the two sides of the Rupununi portal were made using three common metrics: species richness, Shannon diversity and evenness. Significant community differences were found between the Essequibo and Amazon sides of the Rupununi portal. While the Rupununi portal serves as a conduit for some fish, it appears to be functioning as a barrier to dispersal for other species. Our study highlights the significance of the Rupununi portal in shaping fish distributions in this region.