Using the protein sequence of GhPCBER as a probe, six of genes showing high sequence homology were obtained from a genome database of Gossypium hirsutum L.tetraploid cotton with Blastp search. These sequences were used to design primers, and then six genes were isolated from cotton fiber of G. hirsutum L. by using RTPCR technique and designated as GhPCBER1, GhPCBER2, GhPCBER3, GhIFR, GhPLR1 and GhPLR2, respectively. Multiple sequence alignment and phylogenetic tree analysis revealed that all of the 6 proteins belong to PIP subfamily since the conserved motifs and active residues in their sequences. Real time fluorescent quantitative PCR results showed that in addition to GhPLR1, all the other 5 PIP subfamily genes were specifically or preferentially expressed in fiber cells. Of 6 genes, the expression pattern of GhPCBER1, GhPCBER2, GhPCBER3 and GhIFR showed increasing firstly and then decreasing trend during cotton fiber developing stage. The expression level of GhPCBER1 and GhPCBER2 reached a maximum at 21 day post anthesis, and GhIFR and GhPCBER3 reached a maximum at 18 day post anthesis. The expression pattern of GhPLR1 and GhPLR2 showed an ascendant trend with cotton fiber developing. According to the characteristics of gene expression, it is speculated that the PIP subfamily may play an important role in cotton fiber development.