2.25Cr-1Mo steel with high strength at high temperatures and good hydrogen resistance is widely used for power generation boiler material in high temperature and pressure use environments. Following the test evaluation of the ASME Boiler and Pressure Vessel Code, specimens from the base metal of a boiler pipe were found to have impact toughness values of 285 and 21 ft-lb, which are drastically different values. The analysis of the fracture surface of the 21 ft-lb test specimen revealed MnS inclusions, and it was found that cracks initiated at the inclusions. Observation of the cross-section of the crack propagation front revealed that cracks propagated along the ferrite regions and precipitate voids. Inclusions were also found in the 285 ft-lb impact specimen; however, the volume fraction of the inclusions was significantly less than that of the 21 ft-lb specimen. It was also found that the ferrite and carbide content of the 285 ft-lb specimen was less than 21 ft-lb specimen. The reason that the inclusions, ferrite, and carbide content differed in the two adjacent impact test specimens was analyzed. The effects of micro-segregation, such as MnS inclusions on ferrite and carbide, were compared and analyzed.