PHAs are biodegradable and environmentally friendly thermoplastics. The major contributor to PHA production cost is carbon substrate cost, therefore it is desirable to produce PHA from waste/byproducts like Jatropha biodiesel byproduct. This study was done using Jatropha biodiesel byproduct as carbon source, to decrease production cost for PHAs. Total 41 isolates from soil and marine source were able to utilize Jatropha biodiesel-byproduct. Nine bacteria were selected for further studies, which were found positive for Nile red viable colony screening. Two bacterial isolates SM-P-1S and SM-P-3M isolated from soil and marine environment respectively, were found promising for PHA production. PHA accumulation for SM-P-1S and SM-P-3M was 71.8% and 75% PHA/CDW respectively and identified as Bacillus sonorensis and Halomonas hydrothermalis by MTCC. The PHA obtained from SM-P-1S and SM-P-3M was analyzed by FTIR and NMR as Polyhydroxybutyrate (PHB).