Low alloy steels serving for a long time at high temperature, e.g., around 500°C, are very sensitive to temper embrittlement due to segregation of various trace elements at prior austenite grain boundaries and/ or carbide/matrix interfaces. This type of segregation in combination with various environmental effects can adversely affect the fracture resistance and fatigue crack propagation rate with subsequent change in fracture morphology of low alloy steels. This article describes the segregation behavior of various elements in 2.25Cr-1Mo pressure vessel steel investigated by AES, FEG-STEM, SEM, and EDS analyses. As confirmed by AES and FEG-STEM, phosphorus is found to be the main embrittling element for isothermal embrittlement. Sulfur and Mo segregation is only evident after longer embrittlement times. In the stepcooling embrittlement, phosphorus is still found to be the main embrittling element, but heavy segregation of sulfur in some isolated intergranular facets was also observed. For P segregation, a Mo-C-P interaction is observed, while sulfur segregation is attributed to site competition between sulfur and carbon atoms.