Centerline segregation occurs as a positive concentration of alloying elements in the mid-thickness region of continuously cast slab. Depending upon its severity, it may affect mechanical properties and potentially downstream processing such as weldability, particularly for high-strength line pipe. The segregation fraction in continuously cast slabs and corresponding hot-rolled strips was assessed on API 5L grade X65 line pipe steels with different levels of segregation, rated as Mannesmann 2.0 and 1.4. The results showed that the segregation fraction in hot-rolled strip samples was in accordance with that assessed in the cast slabs, and the segregated regions in hot-rolled strip samples were found to be discontinuous. Transverse and z-direction CVN impact tests were conducted on the two strips and the results showed that centerline segregation does have an influence on the Charpy impact properties of line pipe steel. Specimens located at segregated regions exhibited lower Charpy impact toughness and strips rolled from slabs with higher segregation levels are more likely to exhibit greater variability in Charpy impact toughness. The influence of centerline segregation on z-direction Charpy impact toughness is more severe than on transverse Charpy impact toughness. Lower Charpy impact toughness and brittle fracture surface with cleavage facets along with rod-shaped MnS inclusions were observed for the strip rolled from slab with 2.0 segregation rating if the Charpy specimens were located at segregated regions. The influence on Charpy impact toughness can be associated with the pearlite structure at the centerline and level of MnS inclusions.