Scanning electron microscope/electron back-scattering diffraction was used to investigate local misorientation development within an individual α plate of a Ti-6Al-4V alloy with an α lamellar microstructure during hot deformation at 1223 K (950 °C) and a strain rate of 0.1 s−1. The correlation between the local misorientation development and the globularization behavior of α plates during subsequent annealing at 1223 K (950 °C) was discussed. The misorientation profile along an individual α plate showed that not only a continuous and smooth change in orientation but also a discontinuous change in orientation was developed by the hot deformation. We assume that the points where discontinuous change in orientation occurs, Pd, became α/α boundaries and resulted in splitting α plates the annealing. The mean length between adjacent discontinuous points, LI, was determined and compared with the actual mean length of the α plates after hot deformation and subsequent annealing, La, as measured by optical microscopy. The two kinds of length parameters coincided at lower strains, but significant differences were observed at higher strains, i.e., LI was larger than La. Further analysis showed that rotation axes (R.A.s) changed even within regions where orientation changes were continuous. By taking into account the points where the R.A.s changed, Pr and Pd, the mean length between adjacent points, LII, appeared to coincide with La at higher strains. A higher lattice distortion is expected near points Pr at higher strains, which results in the formation of new α/α boundaries in subsequent annealing. Consequently, points Pd already developed by hot deformation were considered to become α/α boundaries and led to splitting α plates in annealing. New α/α boundaries formed at points Pr in subsequent annealing after a higher strain deformation, which led to a splitting of α plates as well.