Sweet sorghum (Sorghum bicolor (Linn.) Moench) has promise as a bioenergy feedstock in China and other countries for its use in the production of ethanol as the result of its high fermentable sugar accumulation in stems. To boost biofuel production and extend its range, we seek to improve its stress tolerance. Proline acts as an osmolyte that accumulates when plants are subjected to abiotic stress. P5CS (Delta1-pyrroline-5-carboxylate synthetase) is a key regulatory enzyme that plays a crucial role in proline biosynthesis. We isolated two closely related P5CS genes from sweet sorghum, designated SbP5CS1 (GenBank accession number: GQ377719) and SbP5CS2 (GenBank accession number: GQ377720), which are located on chromosome 3 and 9 and encode 729 and 716 amino acid polypeptides, respectively. The homology between the two sweet sorghum P5CS genes was 76%. Promoter analysis of the two P5CS genes revealed that both sequences not only contained the expected cis regulatory regions such as TATA and CAAT boxes, but also had many stress response elements. Expression analysis revealed that SbP5CS1 and SbP5CS2 transcripts were up-regulated after treatment of 10-day-old seedlings of sweet sorghum with drought, salt (250mM NaCl) and MeJA (10muM). The expression levels of the both SbP5CS genes were significantly increased after 3-day drought stress. Under high salt treatment, peak SbP5CS1 expression was detected at 4h and 8h for SbP5CS2 in roots, while the trends of expression were nearly identical in leaves. In contrast, under drought and high salt stress, the up-regulated expression of SbP5CS1 was higher than that of SbP5CS2. When the seedlings were exposed to MeJA, rapid transcript induction of SbP5CS1 was detected at 2h in leaves, and the SbP5CS2 expression level increase was detected at 4h post-treatment. SbP5CS1 and SbP5CS2 also show different temporal and spatial expression patterns. SbP5CS2 gene was ubiquitously expressed whereas SbP5CS1 was mainly expressed in mature vegetative and reproductive organs. Proline concentration increased after stress application and was correlated with SbP5CS expression. Our results suggest that the SbP5CS1 and SbP5CS2 are stress inducible genes but might play non-redundant roles in plant development. The two genes could have the potential to be used in improving stress tolerance of sweet sorghum and other bioenergy feedstocks.