A series of poly(3-hydroxybutyrate-co-4-hydroxybutyrate)s [P(3HB-co-4HB)s] with different 4HB content, biosynthesized by Ralstonia eutropha H16 with mixed carbon sources of 4-hydroxybutyric acid (4HBA) and butyric acid, were fractionated by solvent/nonsolvent fractionation into copolyester fractions with different 4HB content and narrower compositional distribution. The fractions obtained were classified into two groups, 3HB- and 4HB-rich P(3HB-co-4HB)s. The thermal properties were investigated for these fractionated copolyesters. With increasing 4HB content, the melting temperature at first decreased while 3HB content was rich, and then increased while 4HB content was rich. The glass transition temperature decreased linearly with increasing 4HB content. The 4HB-rich P(3HB-co-4HB) was found to be immiscible with the 3HB-rich P(3HB-co-4HB), as two glass transitions corresponding to those of respective P(3HB-co-4HB)s were observed by DSC. It was concluded that as-produced bacterial P(3HB-co-4HB) samples used in this study should be considered as immiscible polymer blends.