We identified 575 new NotI landmarks of C57BL/6(B)- and M. spretus (S)-specific, dominant and codominant loci which were segregated in B x S interspecific backcrosses (BSS), using the restriction landmark genomic scanning (RLGS) spot mapping method. All of these loci were visualized on a single RLGS profile which was produced with NotI-PvuII-PstI. These landmarks include 250 newly identified S-specific spots in addition to the previously reported 325 B-specific spots. The S-specific spots were identified by reading full or half intensity, based on the property that the spot intensity of the autoradiographic signal reflected the copy number of an end-labeled restriction landmark. The cumulative map is 1341 cM and it is based upon 985 meiotic events in 72 backcross progeny. This map covers 90% of the total estimated length of the mouse genetic map. This map provides a good tool for the high-speed genome scanning assay in the mouse genome by a single RLGS gel analysis.