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The through-thickness heterogeneous microstructure of thick-section high strength steels is responsible for the significant scatter of properties along the thickness. In this study, in order to identify the critical microstructural features in the fracture behaviour and allow for design optimisation and prediction of structural failure, the through-thickness microstructure of thick-section steels was extensively characterised and quantified. For this purpose, samples were extracted from the top quarter and middle thickness positions, and a combination of techniques including chemical composition analysis, dilatometry, and microscopy was used. The hardness variation through the thickness was analysed via micro-Vickers measurements and the local hardness variation in the middle section was studied via nanoindentation. The middle section presented larger prior austenite grain (PAG) sizes and larger sizes and area fraction of inclusions than the top section. Additionally, cubic inclusions were observed distributed as clusters in the middle, sometimes decorating PAG boundaries. Defects associated with the cubic inclusions or the interface between the matrix and the circular and cubic inclusions were observed in the mid-thickness. Moreover, the middle section presented long interfaces with the most significant hardness gradients due to the presence of hard centreline segregation bands. Hence, the microstructural and nanoindentation analyses indicated the middle section as the most likely area to have the lowest fracture toughness and, therefore, the most unfavourable section for fracture performance of the investigated S690QL high strength steel. The detrimental effect of the middle section was confirmed via CTOD tests where the middle presents lower fracture toughness than the top section.