Infrared (IR) microspectroscopy has the capacity to determine the extent of phase separation in polymer blends. However, a major limitation in the use of this technique has been its reliance on overlapping peaks in the IR spectra to differentiate between polymers of similar chemical compositions in blends. The objective of this study was to evaluate the suitability of deuteration of one mixture component to separate infrared (IR) absorption bands and provide image contrast in phase separated materials. Deuteration of poly(3-hydroxyoctanoate) (PHO) was achieved via microbial biosynthesis using deuterated substrates, and the characteristic C D stretching vibrations provided distinct signals completely separated from the C-H signals of protonated poly(3-hydroxybutyrate) (PHB). Phase separation was observed in 50:50 (% w/w) blends as domains up to 100 pm through the film cross sections, consistent with earlier reports of phase separation observed by scanning electron microscopy (SEM) of freeze-fractured protonated polymer blends. The presence of deuterated phases throughout the film suggests there is some miscibility at smaller length scales, which increased with increasing PHB content. These investigations indicate that biodeuteration combined with IR microspectroscopy represents a useful tool for mapping the phase behavior of polymer blends.