In this paper, a third harmonic was used to investigate microstructural changes in Al6061-T6 due to different fatigue cycles and a relationship between fatigue cycle and third order nonlinearity has been observed. Piezoelectric measurement harmonic generation technique was applied for the specimens with 0%, 55%, 75% and 85% fatigue cycles, respectively. The results shows that the third order harmonics gradually increased up to 55% and rapidly decreased after wards, it was attributed to the behavior of dislocation, dislocation-precipitation interaction and voids with increasing fatigue cycle. Further, it was verified with scanning electron microscope (SEM). We also observed that third order nonlinearity is more sensitive to small change in area of fraction of voids than second order nonlinearity after 55% fatigue life and could be a good candidate to investigate Al6061-T6 specimen with voids.