High strength low alloy steels are widely used in the offshore and marine industries for the manufacturing of fasteners. In this context, the occurrence of hydrogen embrittlement is a risk to the service life of these components. The scope of this work is to evaluate the impact of the steelmaking process on the hydrogen embrittlement susceptibility of AISI 4340 steel through different experimental methodologies. The effects of the presence of segregation bands and inclusions on the hydrogen interaction with the microstructure of the steel were studied through hydrogen electrochemical permeation tests and mechanical characterization. The hydrogen embrittlement susceptibility was investigated by performing uniaxial standard tensile tests with prior hydrogen charging, slow strain rate tests with in-situ hydrogen charging, and incremental step loading tests with notched specimens and in-situ hydrogen charging. Fractographic analyzes were carried out to support the interpretation and discussion of the results. It was observed that continuous casting route results in a microstructure more susceptible to hydrogen embrittlement, due to the presence of coarser segregation bands, with a greater density of impurities and inclusions.