The proximal portion of mouse chromosome 1 harbors a variety of mutant loci that have yet to be characterized at the molecular level. We have constructed a library of genomic DNA fragments from the proximal portion of mouse chromosome 1 by microdissection and microcloning techniques, with the aim of generating genetic markers in close proximity to some of these mutant loci. To facilitate the genetic mapping of 27 microclones from this library, we divided a 56-cM segment of chromosome 1 between the Col3a1 and Ren 1,2 genes into eight intervals defined by anchor loci. Restriction fragment length polymorphisms were determined for each of the microclones and their segregation with the anchor loci was followed in informative animals from a panel of 252 interspecific backcross mice (C57BL/6J x Mus spretus) x C57BL/6J. We were able to assign 26 of 27 (96%) randomly selected microclones to each of the defined chromosome 1 intervals. A total of eight microclones mapped within the large interstitial deletion found in the Spr mouse mutant. Two of these clones were found to be tightly linked to the host resistance locus Bcg and at least one was found to be linked to the neural tube defect mutant splotch. Other clones mapped to intervals containing several other mouse mutants. These novel DNA markers should aid in positional cloning strategies presently employed to identify these mutant loci. These clones should also be useful in the creation of both physical and YAC contiguous maps of the proximal portion of mouse chromosome 1.