To date, only a small percentage of human genes have been cloned and mapped. To facilitate more rapid gene mapping and disease gene isolation, chromosome 5-specific cDNA libraries have been constructed from five sources. DNA sequencing and regional mapping of 205 unique cDNAs indicates that 25 are from known chromosome 5 genes and 138 are from new chromosome 5 genes (a frequency of 79.5%). Sequence complexity estimates indicate that each library contains -20% of the approximately 5000 genes that are believed to reside on chromosome 5. This study more than doubles the number of genes mapped to chromosome 5 and describes an important new tool for disease gene isolation.