The quantitative estimation of strengthening mechanisms of rails’ surface layer is carried out on the basis of regularities and formation mechanisms of structure-phase states revealed by the methods of modern physical materials science. It is performed at different depths of the rail head along the central axis and fillet of differentially quenched 100-meter rails after the extremely long-term operation (gross passed tonnage of 1411 mln tons). A long-term operation of rails is accompanied by the formation of structural constituent gradient consisting of a regular change in the relative content of lamellar pearlite, fractured pearlite, the structure of ferrite-carbide mixture, scalar, and excess dislocation density along the cross-section of the rail head. As the distance to the rail fillet surface decreases, the relative content of metal volume with lamellar pearlite decreases. However, the relative content of metal volume with the presence of the fractured pearlite structure and ferrite-carbide mixture increases. The contributions caused by the matrix lattice friction, intraphase boundaries, dislocation substructure, presence of carbide particles, internal stress fields, solid-solution strengthening, pearlite component of steel structure are estimated. It is shown that the main mechanism of strengthening in the surface layer is due to the interaction of moving dislocations with low-angle boundaries of nanometer dimensional fragments and subgrains. The main dislocation strengthening mechanism in a near-surface layer at a depth of 2-10 mm is due to the interaction of moving dislocations with immobile ones.