Large-insert, unbiased genomic libraries are the key to efficient gene discovery and genome-wide analysis. These libraries are essential for dissecting complicated gene pathways, providing new tools for metabolic engineering. The quality of genomic libraries depends greatly on the cloning systems and vectors used. For example, many viral, microbial, and eukaryotic genomes are refractory to cloning or unstable when cloned into conventional host-vector systems. This cloning bias leads to incomplete libraries and inaccurate analyses. The loss of insert is due to three factors common to most plasmid vectors: vector-driven expression of deleterious sequences; insert-driven transcription into the vector that interferes with vector stability; and unstable secondary structure of insert DNA. We have developed the pSMART series of transcription-free cloning systems to alleviate these problems. The pSMART BAC and fosmid vectors lack an indicator gene and associated promoter, and they have termination signals on either side of the insertion site. As a result, they show much higher stability of inserts in BAC and fosmid libraries. We have also developed the transcription-free pJAZZ vector, a linear cloning plasmid that lacks supercoiling. The pJAZZ system shows much higher stability of inserts containing AT-rich sequences, direct and inverted repeats, and other deleterious DNAs. In addition, we have developed techniques to construct unbiased, randomly-sheared BAC libraries, with average insert sizes of 100~150 kb. The Random Shear BAC libraries show none of the cloning bias or gaps typical of partial restriction libraries. We will discuss the applications of these new technologies for bioenergy, bioprocessing, and environmental bioanalysis.