Twenty microsatellites were generated from a previously characterized lambda gt10 library containing C57BL/6J mouse DNA from a flow-sorted 4:6 Robertsonian chromosome. These sequences were analyzed for size variation between different strains of mice with the polymerase chain reaction (PCR) and mapped by use of either strain distribution patterns (SDPs) in recombinant inbred (RI) strains, or intra- and interspecific backcrosses. Eighty-five percent of the sequences showed allelic variations between different inbred strains of mice and the wild mouse, Mus spretus, and 70% were variant between inbred strains. Eight (62%) of the 13 repeats that have been mapped lie on Chromosomes (Chr) 4 and 6. This approach is an effective way of generating informative markers on specific chromosomes.