Knowledge of the ecology of the fish fauna associated with kelp (primarily Macrocystis pyrifera) forests in Southern Patagonia is scarce, especially in how abiotic and biotic variables influence their structure, diversity, and distribution. This information is important for the management and conservation of this unique ecosystem, which has minimal anthropogenic impacts at present. We analyzed data from 122 quantitative underwater transects conducted within kelp forests at 61 stations from Chile’s southern Patagonian fjords to the Cape Horn and Diego Ramirez archipelagos and the southern tip of Argentina, including the Mitre Peninsula and Isla de los Estados. In total, 25 fish species belonging to 13 families were observed. Multivariate analysis indicated that there are significant differences in fish assemblage structure among locations and wave exposures, which was driven primarily by Patagonotothen sima and Paranotothenia magellanica, which occurred on exposed and semi-exposed stations. P. cornucola was mainly distributed across sheltered stations of the Kawésqar National Park. Temperature, salinity, depth, and kelp density influenced fish assemblage structure, with the highest diversity in areas with the lowest temperature and greater depth at Isla de los Estados. In contrast, species richness, diversity, abundance, and biomass were all lower in areas with high density of the understory kelp Lessonia spp., which might be driven by the absence of P. tessellata, P. squamiceps and P. cornucola, the most important species in terms of occurrence, abundance, and biomass. Our study provides the first broad-scale description of the fish assemblages associated with kelp forests along the southern cone of South America based on non-invasive visual transects, improving our knowledge of the distribution of fish assemblages across several environmental conditions in this vast and little-studied area.