We applied positron annihilation lineshape analysis for non-destructive evaluation of fatigue stored in type 316 stainless steel, mainly used in primary water lines of pressurized water reactors (PWR). Using ⁶⁸Ge as a positron source, an energy spread of annihilation gamma ray peaks from stainless steel specimens was measured. After preparing stress- and strain-controlled fatigue specimens, we investigated the relation between fatigue life and a non-destructive parameter of lineshape analysis defined as the S-parameter and compared the microstructure of the fatigue specimens with the S-parameter. As a result, there was good correlation between the S-parameter and fatigue life; the S-parameter increased with dislocation density monotonically. The relation between the S-parameter and fatigue life in stress-controlled fatigue differed from that in strain-controlled fatigue. The S-parameter increased faster in early stage of the latter than in the former. In stress-controlled fatigue, the change in the S-parameter did not depend on stress amplitude in the range of ratio to yield stress under 0.9. In the strain-controlled fatigue, the change in the S-parameter did not depend on strain amplitude in the range from 0.25 to 0.31%. However, when stress amplitude or strain amplitude became higher, the change in the S-parameter increased largely in the early stage of the fatigue life. We demonstrated systematic data to evaluate the fatigue damage in type 316 stainless steel.