We have constructed a zebrafish yeast artificial chromosome (YAC) library using genomic DNA isolated from the inbred AB zebrafish strain. The average insert size is 470 kb, estimated from analysis of 155 random selected YACs. The library consists of 17,000 clones, providing about a 4.7-fold coverage of zebrafish genome. The YAC clones have been arrayed in individual wells of 96-well microplates and also pooled to permit rapid polymerase chain reaction screening of the entire library. We have also found that the YAC ends can be easily rescued and sequenced from pRML1/pRML2-based mini-YAC clones. Copyright 1998 Academic Press.