Gene silencing has been used widely in gene function studies and crop plant modification. Long hairpin RNA (lhRNA) results in high efficiency of gene silencing; however, constructing multiple lhRNA vectors using traditional approaches is both time consuming and costly. Also, most of the existing approaches are based on sequence-specific cloning of individual sequences and are therefore not suitable for preparing hpRNA libraries from a pool of mixed target sequences. The rolling circle amplification (RCA)-mediated hairpin RNA (RMHR) construction system is suitable for generating hairpin libraries from any gene of interest or pool of genes. Using the RMHR system, a long-hairpin RNA (lhRNA) library is generated from an Arabidopsis cDNA population containing known and unknown genes. Our results indicate that the RMHR technique permits the rapid, efficient, and low-cost preparation of genome-wide lhRNA expression libraries.