Aims: Polyhydroxyalkanoate (PHA) with enhanced physicochemical properties will be ideal for a wide range of practical applications. The incorporation of 3-hydroxy-4-methylvalerate (3H4MV) into the polymer backbone is known to improve the overall properties of the resulting polymer. However, the most suitable micro-organism and PHA synthase that can synthesize this monomer efficiently still remain unknown at present. Therefore, we evaluated the abilities of a locally isolated Chromobacterium sp. USM2 to produce PHA containing 3H4MV. Methods and Results: The ability of Chromobacterium sp. USM2 to synthesize poly(3-hydroxybutyrate-co-3-hydroxy-4-methylvalerate) [P(3HB-co-3H4MV)] was evaluated under different culture conditions. It was found that Chromobacterium sp. USM2 can synthesize P(3HB-co-3H4MV) when glucose and isocaproic acid were fed as carbon source. However, the highest molar fraction of 3H4MV, 22 mol% was detected in Chromobacterium sp. USM2 when isocaproic acid was provided as the sole carbon source. In addition, aeration was identified as a crucial factor in initiating the accumulation of high 3H4MV molar fractions. Conclusions: Chromobacterium sp. USM2 was able to synthesize broad comonomer compositional distribution of P(3HB-co-3H4MV). Significance and Impact of the Study: Compared with Cupriavidus necator and Burkholderia sp., Chromobacterium sp. USM2 was found to have better ability to bioconvert isocaproic acid to form 3H4MV unit.