A total 84 pyrroline5carboxylate synthetase (P5CS) genes were identified from 31 genome sequence available plants by using bioinformatics methods, and their phylogenetic analyses were performed. The results revealed that: (1) P5CS had a large difference in intron length, but there was no significant difference in amino acid length, exon number, isoelectric point, and molecular weight. (2) Due to the occurrence of gene duplication, there were two P5CS in most monocot and dicot plants. (3) Moreover, in both monocot and dicot plants, P5CS1 genes were clustered in one group, while P5CS2 genes were clustered in another group, supporting that P5CS1 and P5CS2 of independent origin and the duplication event occurred before the divergence of monocot and dicot plants. In some plants (such as Medicago truncatula and soybean) there were 3-7 P5CS genes, indicating that the duplication events of P5CS genes also occurred several times after the divergence of monocot and dicot plants, and totally four different evolutionary patterns were revealed. (4) There were two P5CS genes in cassava. Expression analysis revealed that MeP5CS1 and MeP5CS2 were expressed in leaf, petiole, stem, fibrous root and storage root. (5) Of which, MeP5CS1 was highly expressed in leaf, while MeP5CS2 was highly expressed in stem and storage root. Under drought stress, MeP5CS1 was significantly induced in the first fully expanded leaf, while MeP5CS2 was significantly induced in the first fully expanded leaf and bottom leaf. Under cold stress, both MeP5CS1 and MeP5CS2 were significantly induced in different tissues, but they exhibited different expression patterns. These results indicated that MeP5CS1 and MeP5CS2 were involved in the regulation of abiotic stresses (e.g., drought and cold) at the transcriptional level.