The influence of the centerline segregation region (CSR) on the hydrogen embrittlement (HE) of two different API 5L X80 pipeline steel plates was investigated. The novelty of this work was to establish relationships between the CSR, microstructure, and distribution of localized fragile particles on HE susceptibility and on fracture morphology. This work intended to establish a relationship between centerline segregation and HE susceptibility in high-strength low-alloy steels submitted to inhomogeneous transformations. Microscopy, hydrogen permeation, and slow strain rate (SSR) tests were used to investigate hydrogen-related degradation. The solution used on the charging cell of the permeation tests—and on the SSR test cell—was 0.5 mol L−1 H2SO4 + 10 mg L−1 As2O3, and in the oxidation cell, 0.1 M NaOH was used as a solution. The CSR led the thicker plate to present the highest HE index (0.612) in analyses carried out in the mid-thickness; however, the same plate showed the lowest HE index in near-surface tests. The presence of hydrogen changed the fracture morphology from ductile to a brittle and ductile feature; this occurred due to the interaction with localized fragile particles and the significant reduction of the shear stress necessary for the dislocation movement.