In the present study, the effects of the microstructural morphologies of a Ti-6Al-4V (Ti-64) alloy on its fatigue behavior were investigated. Ti-64 bars were subjected to two different thermo-mechanical processing methods. The first sample, referred to as Material-A, had a forged microstructure with the average primary α volume fraction of 44%. The second one, referred to as Material-B, had a hot-rolled microstructure with the average primary α volume fraction of 43%. Fatigue tests were performed on each sample to obtain S-N curves. The microstructure of each sample was observed using an optical microscopy in order to measure the grain sizes of the primary α and secondary α phases. The results of the fatigue tests indicated that Material-B demonstrates better fatigue strength than Material-A. The microstructure of the longitudinal section of each material was also observed to analyze the results of the fatigue tests. The measured diameters and volume fractions of the primary α phases of the two types of materials are similar. On the other hand, the secondary α width of each material is different. It is found that fatigue strength is related to the width of the secondary α phase.