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Abstract Continental margins usually have a high degree of environmental heterogeneity, which, in turn, promotes high benthic biodiversity. The South-Southeast regions concentrate the most well-mapped areas of the Brazilian continental margin regarding seafloor geomorphology and physical oceanography. However, the structure of the soft-sediment benthic fauna of the continental slope is still unknown. Characterization and understanding of the Brazilian continental slope biota are imperative since human activities are increasing in the last decades, especially after the discovery of the pre-salt reservoir in Santos Basin. In this study, we aimed to establish a baseline of the spatial distribution of the benthic macrofaunal communities regarding their latitudinal and bathymetric patterns in the Santos Basin to support future environmental monitoring activities in the region. As part of the Santos Project – The Santos Basin Regional Environmental Characterization (PCR-BS) – coordinated by CENPES/PETROBRAS, a benthic oceanographic cruise was carried out in 2019. Sediment samples were collected using a GOMEX-type box corer in 47 stations distributed in eight transects (400– 2,400 m depth range). In total, 12 additional stations (1,300–2,200 m) were defined to cover an area where oil and gas are exploited. Our results showed that macrofaunal assemblages of the Santos Basin present strong depth zonation related to changes in organic matter input, as well as to temperature, carbonate, and grain size. At local scale, the northern sector stood out for having a higher abundance of macrofauna in the upper slope (400 m) and it probably reflects the oceanographic processes and the organic enrichment caused by the upwelling events that occur at Cabo Frio region. The zonation pattern and the dominance of some polychaete, peracarid crustacean, and bivalve families were similar to other SE Brazilian continental margins. heterogeneity which turn biodiversity SouthSoutheast South Southeast wellmapped mapped oceanography However softsediment soft sediment unknown decades presalt pre salt study region PCRBS PCR BS (PCR-BS CENPESPETROBRAS CENPES PETROBRAS CENPES/PETROBRAS 2019 GOMEXtype GOMEX type 4 400– 400 2400 2 2,40 range. range . range) total 1 1,300–2,200 13002200 300 200 (1,300–2,20 exploited input temperature carbonate size scale (40 polychaete crustacean 201 40 240 2,4 1,300–2,20 1300220 30 20 (1,300–2,2 (4 24 2, 1,300–2,2 130022 3 (1,300–2, ( 1,300–2, 13002 (1,300–2 1,300–2 1300 (1,300– 1,300– 130 (1,300 1,300 13 (1,30 1,30 (1,3 1,3 (1, 1, (1