The superior material properties of Ti-6Al-4V alloy, make it the most desirable material in the aerospace and biomedical industry. This study investigates the impact of microstructural variations on the tensile and fatigue crack growth rate of Ti-6Al-4V alloy. The samples were heat-treated below the β transus temperature in vacuum-sealed conditions, followed by air cooling to obtain three varying microstructures with primary alpha (αp) fractions 54, 31, and 21%. A comprehensive microstructural analysis was conducted to elucidate the influence of different heat treatment temperatures on the microstructure. Fatigue crack growth rate (FCGR) and tensile tests were performed on the heat-treated samples. Lower ductility and better fatigue crack propagation resistance were observed in the samples with lower αp fraction samples due to superior resistance to crack growth provided by the lamellar region consisting of a secondary alpha (αs) phase embedded in a beta (β) matrix.