In the present work, copper mould casting is used to prepare Ti-6Al-4 V alloy in the form of a cylindrical rod of dimension \(\left( {7.5\;{\text{mm }}\emptyset \times 33\;{\text{mm L}}} \right)\) . Microstructural and grain size changes are observed in different regions along the length of the rod sample suggesting the influence of cooling rate during solidification. The present study is undertaken to investigate the microstructural changes in three different regions along the length of the sample. Cooling rates calculated using standard empirical method at different regions are found to be in the range of ~ 1.53 \(\times\) 104–4.3 \(\times\) 104 K/s. The obtained alloy is analysed using micro-indentation to determine the hardness. The present study also probes into the understanding of chemical interaction among the participating elements resulting in bond formation to explain the micro-segregation effect. Grain size variation is observed in the range of 149–411 µm within the same rod sample along the longitudinal direction. This study helps to understand the formation of graded microstructure in Ti-6Al-4 V useful in load-bearing applications.