The microstructure attributes are responsible for the deformation mechanism of material, which induces shear instability primarily in difficult-to-machine material like Ti–6Al–4V. Consequently, the dynamic cutting force yields serrations in the chip morphology. Therefore, microstructure induced shear instability has been investigated in the present work using an analytical tool to unveiled the deformation behavior in correlation with microstructural characteristics (grain sizes, phase fractions, and microhardness) and process parameters: temperature, strain, and strain rate. The combined effect of feed rate and high cutting speed was found to enhance the strain localization phenomena, which leads to a more pronounced cracking, inducing dynamic cutting force. Segmentation frequency and force-frequency correlation imply a significant transition exhibit from the static to dynamic nature of cutting force. The segmentation frequency of the equiaxed microstructure is lowest among the rest at lower cutting speed, revealing the shear instability dependency on the microstructure. The grain size effect restricts the dislocation movement at the higher cutting speed, which led to a larger strain in as-received microstructure followed by equiaxed and fully lamellar microstructure.