Poly(3-hydroxybutyrate) (PHB) is a storage compound synthesized by the bacteria Azotobacter chroococcum. This semi-crystalline polymer is both biodegradable and biocompatible, making it an ideal compound for tissue engineering materials. The key determining factor of the physical and mechanical properties of materials based on PHB is the distribution of amorphous and crystalline phases. Our research aims to further the understanding of how these phases arise and how they interact with each other. Five PHB samples with molecular masses ranging from 384 kdA to 1095 kdA were used for the current study. Raman spectroscopy and surface-free energy calculations were carried out. From our experiments, it was found that samples had similar surface-free energies. Three different boxes of molecular dynamics simulations representing three different hypotheses were set up: a box according to a classical theory of nucleation, a box with shear flow, and a box with heightened hydrophobic interactions. Our simulations show that a box according to the classical theory of nucleation shows the smallest amount of self-organization while the heightened hydrophobic interactions give rise to configurations that resemble reality the most. From our results, we can conclude that for semi-crystalline biological polymers such as PHB, hydrophobic interactions play a significant role in self-organization during the crystallization process.