We have established a new system for chromosome-specific yeast artificial chromosome (YAC) contig construction using restriction landmark genomic scanning (RLGS-based YAC contig mapper). RLGS is a powerful tool for detecting more than 1000 restriction landmarks distributed on an entire genome in one procedure. In this system, RLGS is applied to sorted chromosomes to cover the target chromosome. Using these landmarks as guideposts, chromosome-specific YAC clones are then ordered. In this paper, we report the construction of a map for a human chromosome 21 YAC contig spanning q22.1 using this new approach. Applying RLGS to sorted chromosomes 21 enables detection of approximately 1400 spots (equivalent of 1050 PacI landmarks), covering the entire region of this chromosome. We constructed the 2.5-Mb YAC contig encompassing 21q22.1 with 66 spots (equivalent of 50 PacI landmarks). With this contig map, we could detect two deleted regions and chimerism in the YAC insert DNA. Our results demonstrated the usefulness of this approach for finding DNA alterations of YACs, such as deletions and chimerism.